Method of inducing an antigen-specific immune response by administering a synergistic combination of adjuvants comprising unmethylated CpG-containing nucleic acids and a non-nucleic acid adjuvant

ABSTRACT

The present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant. Such combinations of adjuvants may be used with an antigen or alone. The present invention also relates to methods and products utilizing immunostimulatory oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) for induction of cellular immunity in infants.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/325,193, filed on Jun. 3, 1999, now issued as U.S. Pat. No.6,406,705, which is a continuation in part of U.S. patent applicationSer. No. 09/154,614 filed on Sep. 16, 1998, abandoned, which is aNational Stage filing of PCT/US98/04703, filed on Mar. 10, 1998,claiming priority to U.S. Provisional Patent Application 60/040,376,filed Mar. 10, 1997, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to adjuvants, and in particularto methods and products utilizing a synergistic combination ofoligonucleotides having at least one unmethylated CpG dinucleotide (CpGODN) and a non-nucleic acid adjuvant.

BACKGROUND OF THE INVENTION

Bacterial DNA, but not vertebrate DNA, has direct immunostimulatoryeffects on peripheral blood mononuclear cells (PBMC) in vitro (Krieg etal., 1995). This lymphocyte activation is due to unmethylated CpGdinucleotides, which are present at the expected frequency in bacterialDNA (1/16), but are under-represented (CpG suppression, 1/50 to 1/60)and methylated in vertebrate DNA. Activation may also be triggered byaddition of synthetic oligodeoxynucleotides (ODN) that contain anunmethylated CpG dinucleotide in a particular sequence context. Itappears likely that the rapid immune activation in response to CpG DNAmay have evolved as one component of the innate immune defensemechanisms that recognize structural patterns specific to microbialmolecules.

CpG DNA induces proliferation of almost all (>95%) B cells and increasesimmunoglobulin (Ig) secretion. This B cell activation by CpG DNA is Tcell independent and antigen non-specific. However, B cell activation bylow concentrations of CpG DNA has strong synergy with signals deliveredthrough the B cell antigen receptor for both B cell proliferation and Igsecretion (Krieg et al., 1995). This strong synergy between the B cellsignaling pathways triggered through the B cell antigen receptor and byCpG DNA promotes antigen specific immune responses. In addition to itsdirect effects on B cells, CpG DNA also directly activates monocytes,macrophages, and dendritic cells to secrete a variety of cytokines,including high levels of IL-12 (Klinman et al., 1996; Halpern et al.,1996; Cowdery et al, 1996). These cytokines stimulate natural killer(NK) cells to secrete gamma-interferon (IFN-γ-) and have increased lyticactivity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra;Yamamoto et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces aTh1 like pattern of cytokine production dominated by IL-12 and IFN-γwith little secretion of Th2 cytokines (Klinman et al., 1996).

Hepatitis B virus (HBV) poses a serious world-wide health problem. Thecurrent HBV vaccines are subunit vaccines containing particles of HBVenvelope protein(s) which include several B and T cell epitopes knowncollectively as HBV surface antigen (HBsAg). The HBsAg particles may bepurified from the plasma of chronically infected individuals or morecommonly are produced as recombinant proteins. These vaccines induceantibodies against HBsAg (anti-HBs), which confer protection if presentin titers of at least 10 milli-International Units per milliliter(mIU/ml) (Ellis, 1993). The current subunit vaccines whch contain alum(a Th2 adjuvant), are safe and generally efficacious. They, however,fail to meet all current vaccination needs. For example, earlyvaccination of infants born to chronically infected mothers, as well asothers in endemic areas, drastically reduces the rate of infection, buta significant proportion of these babies will still become chronicallyinfected themselves (Lee et al., 1989; Chen et al., 1996). This couldpossibly be reduced if high titers of anti-HBs antibodies could beinduced earlier and if there were HBV-specific CTL. In addition, thereare certain individuals who fail to respond (non-responders) or do notattain protective levels of immunity (hypo-responders). Finally, thereis an urgent need for an effective treatment for the estimated 350million chronic carriers of HBV and a therapeutic vaccine could meetthis need.

SUMMARY OF THE INVENTION

The present invention relates to methods and products for inducing animmune response. The invention is useful in one aspect as a method ofinducing an antigen specific immune response in a subject. The methodincludes the steps of administering to the subject in order to induce anantigen specific immune response an antigen and a combination ofadjuvants, wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant, and wherein the combinationof adjuvants is administered in an effective amount for inducing asynergistic adjuvant response. In one embodiment the subject is aninfant.

The CpG oligonucleotide and the non-nucleic acid adjuvant may beadministered with any or all of the administrations of antigen. Forinstance the combination of adjuvants may be administered with a primingdose of antigen. In another embodiment the combination of adjuvants isadministered with a boost dose of antigen. In some embodiments thesubject is administered a priming dose of antigen and oligonucleotidecontaining at least one unmethylated CpG dinucleotide before the boostdose. In yet other embodiments the subject is administered a boost doseof antigen and oligonucleotide containing at least one unmethylated CpGdinucleotide after the priming dose.

The antigen may be any type of antigen known in the art. For example,the antigen may be selected from the group consisting of peptides,polypeptides, cells, cell extracts, polysaccharides, polysaccharideconjugates, lipids, glycolipids and carbohydrates. Antigens may be givenin a crude, purified or recombinant form and polypeptide/peptideantigens, including peptide mimics of polysaccharides, may also beencoded within nucleic acids. Antigens may be derived from an infectiouspathogen such as a virus, bacterium, fungus or parasite, or the antigenmay be a tumor antigen, or the antigen may be an allergen.

According to another aspect of the invention a method of inducing a Th1immune response in a subject is provided. The method includes the stepof administering to the subject in order to induce a Th1 immune responsea combination of adjuvants, wherein the combination of adjuvantsincludes at least one oligonucleotide containing at least oneunmethylated CpG dinucleotide and at least one non-nucleic acidadjuvant, and wherein the combination of adjuvants is administered in aneffective amount for inducing a Th1 immune response. In one embodimentthe combination of adjuvants is administered simultaneously. In anotherembodiment the combination of adjuvants is administered sequentially. Insome embodiments the combination of adjuvants is administered in aneffective amount for inducing a synergistic Th1 immune response. Inanother aspect, the same method is performed but the subject is aninfant and the Th1 response can be induced using CpG DNA alone, or CpGDNA in combination with a non-nucleic acid adjuvant at the same ordifferent site, at the same or different time.

The invention in other aspects is a composition of a synergisticcombination of adjuvants. The composition includes an effective amountfor inducing a synergistic adjuvant response of a combination ofadjuvants, wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant. The composition may alsoinclude at least one antigen, which may be selected from the groupconsisting of peptides, polypeptides, cells, cell extracts,polysaccharides, polysaccharide conjugates, lipids, glycolipids andcarbohydrates. Antigens may be given in a crude, purified or recombinantform and polypeptide/peptide antigens, including peptide mimics ofpolysaccharides, may also be encoded within nucleic acids. Antigens maybe derived from an infectious pathogen such as a virus, bacterium,fungus or parasite, or the antigen may be a tumor antigen, or theantigen may be an allergen.

According to other aspects the invention includes a method forimmunizing an infant. The method involves the step of administering toan infant an antigen and an oligonucleotide containing at least oneunmethylated CpG dinucleotide and at least one non-nucleic acid adjuvantin an effective amount for inducing cell mediated immunity or Th1-likeresponses in the infant. The method may also involve the step ofadministering at least one non-nucleic acid adjuvant.

The CpG oligonucleotide may be administered with any or all of theadministrations of antigen. For instance the CpG oligonucleotide or thecombination of adjuvants may be administered with a priming dose ofantigen. In another embodiment the CpG oligonucleotide or thecombination of adjuvants is administered with a boost dose of antigen.In some embodiments the subject is administered a priming dose ofantigen and oligonucleotide containing at least one unmethylated CpGdinucleotide before the boost dose. In yet other embodiments the subjectis administered a boost dose of antigen and oligonucleotide containingat least one unmethylated CpG dinucleotide after the priming dose.

The invention in other aspects includes a method of inducing a strongerTh1 immune response in a subject being treated with a non-nucleic acidadjuvant. The method involves the steps of administering to a subjectreceiving an antigen and at least one non-nucleic acid adjuvant and atleast one oligonucleotide containing at least one unmethylated CpGdinucleotide in order to induce a stronger Th1 immune response thaneither the adjuvant or oligonucleotide produces alone.

The invention in other aspects include a method of inducing anon-antigen-specific Th1-type immune response, including Th1 cytokinessuch as IL-12 and IFN-γ, for temporary protection against variouspathogens including viruses, bacteria, parasites and fungi. The methodinvolves the steps of administering to a subject at least onenon-nucleic acid adjuvant and at least one oligonucleotide containing atleast one unmethylated CpG dinucleotide in order to induce a Th1 innateimmune response. For longer term protection, these adjuvants may beadministered more than once. In another embodiment, CpG DNA may be usedalone at one or more of the administrations.

In each of the above described embodiments a CpG oligonucleotide is usedas an adjuvant. The oligonucleotide in one embodiment contains at leastone unmethylated CpG dinucleotide having a sequence including at leastthe following formula:5′ X₁X₂CGX₃X₄ 3′wherein C and G are unmethylated, wherein X₁X₂ and X₃X₄ are nucleotides.In one embodiment the 5′ X₁X₂CGX₃X₄ 3′ sequence is a non-palindromicsequence.

The oligonucleotide may be modified. For instance, in some embodimentsat least one nucleotide has a phosphate backbone modification. Thephosphate backbone modification may be a phosphorothioate orphosphorodithioate modification. In some embodiments the phosphatebackbone modification occurs on the 5′ side of the oligonucleotide orthe 3′ side of the oligonucleotide.

The oligonucleotide may be any size. Preferably the oligonucleotide has8 to 100 nucleotides. In other embodiments the oligonucleotide is 8 to40 nucleotides in length.

In some embodiments X₁X₂ are nucleotides selected from the groupconsisting of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT,and TpG; and X₃X₄ are nucleotides selected from the group consisting of:TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.Preferably X₁X₂ are GpA or GpT and X₃X₄ are TpT. In other preferredembodiments X₁ or X₂ or both are purines and X₃ or X₄ or both arepyrimidines or X₁X₂ are GpA and X₃ or X₄ or both are pyrimidines. In oneembodiment X₂ is a T and X₃ is a pyrimidine. The oligonucleotide may beisolated or synthetic.

The invention also includes the use of a non-nucleic acid adjuvant insome aspects. The non-nucleic acid adjuvant in some embodiments is anadjuvant that creates a depo effect, an immune stimulating adjuvant, oran adjuvant that creates a depo effect and stimulates the immune system.Preferably the adjuvant that creates a depo effect is selected from thegroup consisting of alum (e.g., aluminum hydroxide, aluminum phosphate)emulsion based formulations including mineral oil, non-mineral oil,water-in-oil or oil-in-water emulsions, such as the Seppic ISA series ofMontanide adjuvants; MF-59; and PROVAX. In some embodiments the immunestimulating adjuvant is selected from the group consiting of saponinspurified from the bark of the Q. saponaria tree, such as QS21;poly[di(carboxylatophenoxy)phosphazene (PCPP) derivatives oflipopolysaccharides such as monophosphorlyl lipid (MPL), muramyldipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM-174; andLeishmania elongation factor. In one embodiment the adjuvant thatcreates a depo effect and stimulates the immune system is selected fromthe group consiting of ISCOMS; SB-AS2; SB-AS4; non-ionic blockcopolymers that form micelles such as CRL 1005; and Syntex AdjuvantFormulation.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 has two graphs illustrating humoral and cytotoxic T-lymphocyte(CTL) responses in adult BALB/c mice immunized with 1 μg recombinantHBsAg protein alone, adsorbed onto alum (25 mg Al³⁺/mg HBsAg), with 100μg of immunostimulatory CpG ODN 1826, or with both alum and CpG ODN.Left panel: Each point represents the group mean (n=10) for titers ofanti-HBs (total IgG) as determined in triplicate by end-point dilutionELISA assay. End-point titers were defined as the highest plasmadilution that resulted in an absorbance value (OD 450) two times greaterthan that of control non-immune plasma with a cut-off value of 0.05.Right panel: Each point represents the mean % specific lysis at theindicated effector: target (E:T) cell ratio in a chromium release assaywith HBsAg-expressing cells as targets.

FIG. 2 is a graph illustrating humoral responses in adult BALB/c miceimmunized with 1 μg recombinant HBsAg protein, with or without alum, andwith 0, 0.1, 1, 10, 100 or 500 μg of CpG ODN 1826 added. Each pointrepresents the group mean (n=10) for anti-HBs titers (total IgG) asdetermined by end-point dilution ELISA assay.

FIG. 3 is a graph illustrating humoral responses in adult BALB/c miceimmunized with 1 μg recombinant HBsAg protein, with or without alum, andwith one of several different oligonucleotides (ODN, 10 μg). The ODNwere made with a natural phosphodiester backbone (O), syntheticphosphorothioate backbone (S) or a chimeric of phosphodiester centerregions and phosphorothioate ends (SOS). Most of the ODN contained 1-3CpG motifs but some of the ODN were non-CpG controls (1911, 1982, 2041).Each point represents the group mean (n=5) for anti-HBs titers (totalIgG) as determined by end-point dilution ELISA assay.

FIG. 4 is a graph of CTL responses in adult BALB/c mice immunized with 1μg recombinant HBsAg protein with alum (25 mg Al³⁺/mg HBs/Ag), with 10μg of CpG ODN 1826, or with both alum and CpG ODN. Some animals wereboosted with the same or a different formulation after 8 weeks. Eachpoint represents the group mean (n=5) for % specific lysis ofHBsAg-expressing target cell at various effector:target (E:T) cellratios.

FIG. 5 is a graph of humoral responses in BALB/c mice immunized withHBsAg (1 μg) without adjuvant or with various adjuvants alone or incombination. The adjuvants were: alum (25 mg Al³⁺/mg HBs/Ag), with CpGDNA (10 μg CpG ODN 1826), monophosphoryl lipid A (MPL, 50 μg) andFreund's complete adjuvant (mixed 1:1 v/v with HBsAg solution). Eachpoint represents the group mean (n=10) for anti-HBS titers (total IgG)as determined by end-point dilution ELISA assay 4 weeks afterimmunization.

FIG. 6 is a bar graph depicting the amount of total IgG (end-point ELISAtiter) produced at 4 weeks in BALB/c mice immunized with 1 μg of HBsAgwith or without CpG and/or IFA (mineral oil mixed 1:1 v/v) or CFA(complete Freund's adjuvant mixed 1:1 v/v). The numbers above each barindicate the IgG2a:IgG1 ratio, with a number in excess of 1 indicating amore Th1-like response.

FIG. 7 is a bar graph depicting the amount of total IgG produced at 4weeks in BALB/c mice immunized with 1 μg of HBsAg with or without CpGand/or MPL (monophosphoryl lipid A, 50 μg) or alum. The numbers aboveeach bar indicate the IgG2a:IgG1 ratio, with a number in excess of 1indicating a more Th1-like response.

FIG. 8 is a graph of the percent of young BALB/c mice that seroconverted(end-point dilution titer >100) after immunization at <1, 3, 7 or 14days of age. Mice were immunized with 10 μg HBsAg-expressing DNA vaccine(pCMV-S), or with recombinant HBsAg (1 μg) with alum (25 mg Al3+/mgHBsAg), CpG ODN 1826 (10 μg) or both alum and CpG ODN. Each pointrepresents the proportion of mice responding, the numbers above the barsshow the number of responding over the total number immunized.

FIG. 9 has two graphs illustrating humoral and cytotoxic T-lymphocyte(CTL) responses in BALB/c mice immunized at 7 days of age with a DNAvaccine (1 μg pCMV-S), or with 1 μg recombinant HBsAg protein alone,adsorbed onto alum (25 mg Al³⁺/mg HBsAg), with 100 μg ofimmunostimulatory CpG ODN 1826, or with both alum and CpG ODN. Upperpanel: Each point represents the group mean of animals thatseroconverted (see FIG. 8 for numbers of animals) for titers of anti-HBs(total IgG) as determined in triplicate by end-point dilution ELISAassay. End-point titers were defined as the highest plasma dilution thatresulted in an absorbance value (OD 450) two times greater than that ofcontrol non-immune plasma with a cut-off value of 0.05. Lower panel:Each point represents the mean % specific lysis at the indicatedeffector: target (E:T) cell ratio in a chromium release assay withHBsAg-expressing cells as targets.

FIG. 10 is a bar graph illustrating humoral responses in neonatal BALB/cmice at 8 weeks after immunization (at 7 days of age) with 1 μgrecombinant HBsAg protein with alum (25 mg Al³⁺/mg HBsAg), with 10 μg ofCpG ODN 1826, or with both alum and CpG ODN. Each point represents thegroup mean (see FIG. 8 for numbers of animals) for anti-HBs titers (IgG1and IgG2a isotypes) as determined by end-point dilution ELISA assay.IgG1 antibodies indicate a Th2-biased response whereas IgG2a antibodiesare indicative of a Th1-type response.

FIG. 11 is a graph of humoral responses in juvenile Cynomolgus monkeysimmunized with Engerix-B vaccine (10 μg recombinant HBsAg protein withalum, SmithKline Beecham biologicals, Rixensart, BE) or with Engerix-Bplus 500 μg of CpG ODN 1968. Each point represents the group mean (n=5)for anti-HBs titers in milli-International units/ml (mIU/ml). A titer of10 mIU/ml is considered protective in humans.

FIG. 12 is a bar graph depicting titers of antibodies against HBsAg(anti-HBs) in millilnternational Units per millilitre (mIU/ml) inorangutans immunized with 10 μg HBsAg with alum (like the HBV commercialvaccine), CpG oligonucleotides (CpG ODN 2006, 1 mg) or both alum and CpGODN. The numbers above the bars show the number of animals withseroconversion (upper numbers, >1 mIU/ml) or with seroprotection (lowernumbers, >10 mIU/ml) over the total number of animals immunized. A titerof 10 mIU/ml is considered sufficient to protect humans and great apesagainst infection.

DETAILED DESCRIPTION OF THE INVENTION

The invention in one aspect is based on the discovery that formulationscontaining combinations of immunostimulatory CpG oligonucleotides andnon-nucleic acid adjuvants synergistically enhance immune responses to agiven antigen. Different non-nucleic acid adjuvants used in combinationin the prior art have different affects on immune system activation.Some combinations of adjuvants produce an antigen-specific response thatis no better than the best of the individual components and somecombinations even produce lower antigen specific responses than with theindividual adjuvants used alone. In Gordon et al., for instance, whenhumans were immunized with C terminal recombinant malariacircumsporozite antigen with alum alone or alum in combination withmonophosphoryl lipid A (MPL), the subjects receiving alum alonedeveloped higher antigen specific antibodies at several time points thansubjects receiving the combination of adjuvants.

It has been discovered according to the invention that the combinationof immunostimulatory CpG oligonucleotides and alum, MPL and otheradjuvants results in a synergistic immune response. Compared with therecombinant hepatitis B surface antigen (HBsAg) protein vaccine alone,addition of alum increases the level of antibodies in mice against HBsAg(anti-HBs) about 7-fold whereas addition of CpG ODN increases them32-fold. When CpG ODN and alum are used together, a 500-1000 timeshigher level of anti-HBs was observed, indicating a strong synergisticresponse. Additionally, it was found according to the invention thatimmunization with HBsAg and alum resulted in a strong Th2-type responsewith almost all IgG being of the IgG1 isotype. CpG ODN induced a highproportion of IgG2a, indicative of a Th1-type response, even in thepresence of alum. Furthermore, it was discovered according to theinvention that in very young mice (7 day old), immune responses wereinduced by HBsAg with alum and CpG ODN but not with alum or CpG ODNalone. The antibodies produced with CpG ODN were predominantly of theIgG2a isotype, indicating a strong Th1-type response. This is remarkableconsidering the strong Th2 bias of the neonatal immune system and theknown difficulty in inducing Th1 responses at such a young age. Th1responses are preferable in some instances since they are associatedwith IgG2a antibodies that have better neutralization and opsonizationcapabilities than Th2-type antibodies. As well, Th1 responses areassociated with cytotoxic T lymphocytes (CTL) that can attack and killvirus-infected cells. Indeed, CpG ODN, alone or in combination with aluminduced good CTL activity in both adult and neonatal mice. These studiesdemonstrate that the addition of CpG ODN to protein or DNA vaccines incombination with other adjuvants is a valid new adjuvant approach toimprove efficacy.

Thus in one aspect the invention is a method of inducing an antigenspecific immune response in a subject. The method includes the step ofadministering to the subject in order to induce an antigen specificimmune response an antigen and a combination of adjuvants, wherein thecombination of adjuvants includes at least one oligonucleotidecontaining at least one unmethylated CpG dinucleotide and at least onenon-nucleic acid adjuvant, and wherein the combination of adjuvants isadministered in an effective amount for inducing a synergistic adjuvantresponse.

The synergistic combination of adjuvants is particularly useful as aprophylactic vaccine for the treatment of a subject at risk ofdeveloping an infection with an infectious organism or a cancer in whicha specific cancer antigen has been identified or an allergy where theallergen is known. The combination of adjuvants can also be givenwithout the antigen or allergen for shorter term protection againstinfection, allergy or cancer, and in this case repeated doses will allowlonger term protection. A “subject at risk” as used herein is a subjectwho has any risk of exposure to an infection causing pathogen or acancer or an allergen or a risk of developing cancer. For instance, asubject at risk may be a subject who is planning to travel to an areawhere a particular type of infectious agent is found or it may be asubject who through lifestyle or medical procedures is exposed to bodilyfluids which may contain infectious organisms or even any subject livingin an area that an infectious organism or an allergen has beenidentified. Subjects at risk of developing infection also includegeneral populations to which a medical agency recommends vaccinationwith a particular infectious organism antigen. If the antigen is anallergen and the subject develops allergic responses to that particularantigen and the subject is exposed to the antigen, i.e., during pollenseason, then that subject is at risk of exposure to the antigen.

There is a need for a prophylactic vaccine that can induce protectiveimmunity against many infectious pathogens more quickly and with fewerdoses than traditional vaccines can provide. For instance, fewer than20% of healthy individuals attain protective levels of anti-hepatitis B(HB) antibodies (10 mIU/ml) after a single dose of subunit hepatitis BVaccine (HBV) vaccine and only 60-70% reach this level after two doses.Thus, three doses (usually given at 0, 1 and 6 months) are required toseroconvert >90% of vaccinated individuals. The three dose regime isfrequently not completed owing to poor patient compliance, and inendemic areas, protective levels may not be induced quickly enough. Themethods of the invention are particularly useful as prophylactictreatments because they induce higher levels of antibodies than can beachieved with traditional vaccines and can be administered as fewertotal doses.

Additionally between 5 and 10% of individuals are non-responders orhypo-responders to the subunit HBsAg vaccine. This may be MHC-restricted(Kruskall et al., 1992) and is thought to result from a failure torecognize T-helper epitopes. In certain immunocompromised individuals(e.g., kidney dialysis patients, alcoholics) the rate of non-responsecan approach 50%. As set forth in the Examples below, alum plus CpG ODNgave higher anti-HBs titers than alum alone in a strain of mice whichhas MHC-restricted hypo-responsiveness to HBsAg, thought to result in afailure to recognize T-helper epitopes. CpG ODN also overcamenon-response in mice genetically incapable of providing T-help owing toan absence of class II MHC. Similar results were obtained in orangutansat risk of becoming infected with hepatitis B. It was found thatorangutans are hyporesponders to the classical alum-adjuvanted vaccinewith less than 10% achieving seroprotection after 2 doses, but thatnearly 100% of animals responded with use of CpG oligonucleotides aloneor combined with alum. The synergistic response was evident becauseantibody titers were much higher with CpG ODN plus alum than with CpGODN alone or alum alone and were more than additive. These resultssupport the proposition that CpG ODN drives the T cell independentactivation of B cells. Thus in addition to providing a more effectiveand easier vaccination protocol the synergistic combination of adjuvantscan be used to reduce the percentage of non-responders andhypo-responders.

A subject at risk of developing a cancer is one who is who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission. When asubject at risk of developing a cancer is treated with an antigenspecific for the type of cancer to which the subject is at risk ofdeveloping and an adjuvant and a CpG oligonucleotide the subject may beable to kill of the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In addition to the use of the combination of adjuvants for prophylactictreatment, the invention also encompasses the use of the combination forthe immunotherapeutic treatment of a subject having an infection, anallergy or a cancer. A “subject having an infection” is a subject thathas been exposed to an infectious pathogen and has acute or chronicdetectable levels of the pathogen in the body. The combination ofadjuvants can be used with an antigen to mount an antigen specificimmune response that is capable of reducing the level of or eradicatingthe infectious pathogen. An infectious disease, as used herein, is adisease arising from the presence of a foreign microorganism in thebody.

Many types of infectious pathogens do not have any effective treatmentsand chronic presence of the pathogen can result in significant damage.For instance, the HBV virus is itself non-pathogenic but with chronicinfection the partially developed immune response causes inflammatorychanges that eventually leads to cirrhosis and increased risk ofhepatocellular carcinoma. An estimated one million people die each yearfrom HBV-related liver disease. Persistent HBV infection of the liverresults when acute infection fails to launch an appropriate immuneresponse to clear the virus. Such chronic carriers have circulatingHBsAg “e” soluble form of the HBV core antigen (HBeAg) without specificimmunity. It is thought that the absence of HBV-specific T-cells,including CTL may contribute to the establishment and maintenance of thechronic carrier state. Indeed, many previously infected individuals,even years after clinical and serological recovery, have traces of HBVin their blood and HBV-specific CTL that express activation markersindicative of recent contact with antigen (Rehermann et al., 1996).These results suggest that sterilizing immunity may not occur after HBVinfection and that chronic activation of HBV-specific CD4+ and CD8+T-cells is responsible for keeping the virus under control.

There is currently no cure for the HBV chronic infection. Interferon isused currently but this cures only 10-20% of treated individuals(Niederau et al., 1996). Anti-viral drugs (e.g., lamivudine) can reducecirculating virus to undetectable levels, however these return topretreatment levels if the drug is stopped. Each of these types oftreatment is also expensive and has certain undesirable side-effects.Thus the synergistic combination of adjuvants which induces potent Th1responses, including CTL, is useful for treating a subject having aninfection such as HBV.

A “subject having an allergy” is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An “allergy”refers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, conjunctivitis, hay fever, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Currently, allergic diseases are generally treated eithersymptomatically with antihistimines for example or immunotherapeuticallyby the injection of small doses of antigen followed by subsequentincreasing dosage of antigen. Symptomatic treatment offers onlytemporary relief. Immunotherapy is believed to induce tolerance to theallergen to prevent further allergic reactions. This approach, however,takes several years to be effective and is associated with the risk ofside effects such as anaphylactic response. The methods of the inventionavoid these problems.

Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by unmethylated CpGoligonucleotides are predominantly of a class called “Th1” whichincludes IL-12 and IFN-γ. In contrast, Th2 immune response areassociated with the production of IL-4, IL-5 and IL-10. Th1 responsesinclude both cell-mediated responses (including cytotoxic T-cells) andantibodies, whereas Th2 responses are associated only with antibodies.The antibodies with a Th1 response are of isotypes (e.g. IgG2a) thathave better neutralizing and opsonizing capabilities than those of Th2isotypes (e.g. IgE that mediates allergic responses). In general, itappears that allergic diseases are mediated by Th2 type immune responsesand protective immune responses by Th1 immune response althoughexaggerated Th1 responses may be also associated with autoimmunediseases.

Th2 cytokines, especially IL-4 and IL-5 are elevated in the airways ofasthmatic subjects. These cytokines promote important aspects of theasthmatic inflammatory response, including IgE isotype switching,eosinophil chemotaxis and activation and mast cell growth. Th1cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2clones and production of Th2 cytokines. “Asthma” refers to a disorder ofthe respiratory system characterized by inflammation, narrowing of theairways and increased reactivity of the airways to inhaled agents.Asthma is frequently, although not exclusively associated with atopic orallergic symptoms.

Based on the ability of the CpG oligonucleotides to shift the immuneresponse in a subject from a Th2 (which is associated with production ofIgE antibodies and allergy) to a Th1 response (which is protectiveagainst allergic reactions), an effective dose of a CpG oligonucleotidecan be administered to a subject to treat or prevent an allergy.

Since Th1 responses are even more potent with CpG DNA combined withnon-nucleic acid adjuvants, the combination of adjuvants of the presentinvention will have significant therapeutic utility in the treatment ofallergic conditions such as asthma. Such combinations of adjuvants couldbe used alone or in combination with allergens.

A “subject having a cancer” is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A “subject” shall mean a human or vertebrate animal including but notlimited to a dog, cat, horse, cow, pig, sheep, goat, chicken, primate,(e.g., monkey), fish (aquaculture species e.g. salmon, trout and othersalmonids), rat, and mouse.

The subject is administered a combination of adjuvants, wherein thecombination of adjuvants includes at least one oligonucleotidecontaining at least one unmethylated CpG dinucleotide and at least onenon-nucleic acid adjuvant. An oligonucleotide containing at least oneunmethylated CpG dinucleotide is a nucleic acid molecule which containsan umnethylated cytosine-guanine dinucleotide sequence (i.e. “CpG DNA”or DNA containing a 5′ cytosine followed by 3′ guanosine and linked by aphosphate bond) and activates the immune system. The CpGoligonucleotides can be double-stranded or single-stranded. Generally,double-stranded molecules are more stable in vivo, while single-strandedmolecules have increased immune activity. The CpG oligonucleotides orcombination of adjuvants can be used with or without antigen.

The terms “nucleic acid” and “oligonucleotide” are used interchangeablyto mean multiple nucleotides (i.e. molecules comprising a sugar (e.g.ribose or deoxyribose) linked to a phosphate group and to anexchangeable organic base, which is either a substituted pyrimidine(e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine(e.g. adenine (A) or guanine (G)). As used herein, the terms refer tooligoribonucleotides as well as oligodeoxyribonucleotides. The termsshall also include polynucleosides (i.e. a polynucleotide minus thephosphate) and any other organic base containing polymer. Nucleic acidmolecules can be obtained from existing nucleic acid sources (e.g.genomic or cDNA), but are preferably synthetic (e.g. produced byoligonucleotide synthesis). The entire CpG oligonucleotide can beunmethylated or portions may be unmethylated but at least the C of the5′ CG 3′ must be unmethylated.

In another embodiment the invention provides an isolated CpGoligonucleotide represented by at least the formula:5′ N₁X₁CGX₂N₂ 3′wherein at least one nucleotide separates consecutive CpGs; X₁ isadenine, guanine, or thymine; X₂ is cytosine, adenine, or thymine; N isany nucleotide and N₁ and N₂ are nucleic acid sequences composed of fromabout 0-25 N's each.

In another embodiment the invention provides an isolated CpGoligonucleotide represented by at least the formula:5′ N₁X₁X₂CGX₃X₄N₂ 3′wherein at least one nucleotide separates consecutive CpGs; X₁X₂ arenucleotides selected from the group consisting of: GpT, GpG, GpA, ApA,ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X₃X₄ are nucleotidesselected from the group consisting of: TpT, CpT, ApT, TpG, ApG, CpG,TpC, ApC, CpC, TpA, ApA, and CpA; N is any nucleotide and N₁ and N₂ arenucleic acid sequences composed of from about 0-25 N's each. PreferablyX₁X₂ are GpA or GpT and X₃X₄ are TpT. In other preferred embodiments X₁or X₂ or both are purines and X₃ or X₄ or both are pyrimidines or X₁X₂are GpA and X₃ or X₄ or both are pyrimidines. In a preferred embodimentN₁ and N₂ of the nucleic acid do not contain a CCGG or CGCG quadmer ormore than one CCG or CGG trimer. The effect of a CCGG or CGCG quadmer ormore than one CCG or CGG trimer depends in part on the status of theoligonucleotide backbone. For instance, if the oligonucleotide has aphosphodiester backbone or a chimeric backbone the inclusion of thesesequences in the oligonucleotide will only have minimal if any affect onthe biological activity of the oligonucleotide. If the backbone iscompletely phosphorothioate or significantly phosphorothioate then theinclusion of these sequences may have more influence on the biologicalactivity or the kinetics of the biological activity. In anotherpreferred embodiment the CpG oligonucleotide has the sequence 5′TCN₁TX₁X₂CGX₃X₄3′. (SEQ ID NO.:99).

Preferably the CpG oligonucleotides of the invention include X₁X₂selected from the group consisting of GpT, GpG, GpA and ApA and X₃X₄ isselected from the group consisting of TpT, CpT and GpT. For facilitatinguptake into cells, CpG containing oligonucleotides are preferably in therange of 8 to 30 bases in length. However, nucleic acids of any sizegreater than 8 nucleotides (even many kb long) are capable of inducingan immune response according to the invention if sufficientimmunostimulatory motifs are present, since larger nucleic acids aredegraded into oligonucleotides inside of cells. Preferred syntheticoligonucleotides do not include a CCGG or CGCG quadmer or more than oneCCG or CGG trimer at or near the 5′ and/or 3′ terminals. Stabilizedoligonucleotides, where the oligonucleotide incorporates a phosphatebackbone modification, as discussed in more detail below are alsopreferred. The modification may be, for example, a phosphorothioate orphosphorodithioate modification. Preferably, the phosphate backbonemodification occurs at the 5′ end of the nucleic acid for example, atthe first two nucleotides of the 5′ end of the oligonucleotide. Further,the phosphate backbone modification may occur at the 3′ end of thenucleic acid for example, at the last five nucleotides of the 3′ end ofthe nucleic acid. Alternatively the oligonucleotide may be completely orpartially modified.

Preferably the CpG oligonucleotide is in the range of between 8 and 100and more preferably between 8 and 30 nucleotides in size. Alternatively,CpG oligonucleotides can be produced on a large scale in plasmids. Thesemay be administered in plasmid form or alternatively they can bedegraded into oligonucleotides.

The CpG oligonucleotide and immunopotentiating cytokine may be directlyadministered to the subject or may be administered in conjunction with anucleic acid delivery complex. A “nucleic acid/cytokine deliverycomplex” shall mean a nucleic acid molecule and/or cytokine associatedwith (e.g. ionically or covalently bound to; or encapsulated within) atargeting means (e.g. a molecule that results in higher affinity bindingto target cell (e.g. dendritic cell surfaces and/or increased cellularuptake by target cells). Examples of nucleic acid/cytokine deliverycomplexes include nucleic acids/cytokines associated with: a sterol(e.g. cholesterol), a lipid (e.g. a cationic lipid, virosome orliposome), or a target cell specific binding agent (e.g. a ligandrecognized by target cell specific receptor). Preferred complexes shouldbe sufficiently stable in vivo to prevent significant uncoupling priorto internalization by the target cell. However, the complex should becleavable under appropriate conditions within the cell so that thenucleic acid/cytokine is released in a functional form.

“Palindromic sequence” shall mean an inverted repeat (i.e. a sequencesuch as ABCDEE′D′C′B′A′ in which A and A′ are bases capable of formingthe usual Watson-Crick base pairs. In vivo, such sequences may formdouble-stranded structures. In one embodiment the CpG oligonucleotidecontains a palindromic sequence. A palindromic sequence used in thiscontext refers to a palindrome in which the CpG is part of thepalindrome, and preferably is the center of the palindrome. In anotherembodiment the CpG oligonucleotide is free of a palindrome. A CpGoligonucleotide that is free of a palindrome is one in which the CpGdinucleotide is not part of a palindrome. Such an oligonucleotide mayinclude a palindrome in which the CpG is not part of the palindrome.

A “stabilized nucleic acid molecule” shall mean a nucleic acid moleculethat is relatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Stabilization can be a function of length or secondarystructure. Unmethylated CpG oligonucleotides that are tens to hundredsof kbs long are relatively resistant to in vivo degradation,particularly when in a double-stranded closed-circular form (i.e., aplamid). For shorter CpG oligonucleotides, secondary structure canstabilize and increase their effect. For example, if the 3′ end of anoligonucleotide has self-complementarity to an upstream region, so thatit can fold back and form a sort of stem loop structure, then theoligonucleotide becomes stabilized and therefore exhibits more activity.

Preferred stabilized oligonucleotides of the instant invention have amodified backbone. It has been demonstrated that modification of theoligonucleotide backbone provides enhanced activity of the CpGoligonucleotides when administered in vivo. CpG constructs, including atleast two phosphorothioate linkages at the 5′ end of the oligonucleotidein multiple phosphorothioate linkages at the 3′ end, preferably 5,provides maximal activity and protected the oligonucleotide fromdegradation by intracellular exo- and endo-nucleases. Other modifiedoligonucleotides include phosphodiester modified oligonucleotide,combinations of phosphodiester and phosphorothioate oligonucleotide,methylphosphonate, methylphosphorothioate, phosphorodithioate, andcombinations thereof. Each of these combinations and their particulareffects on immune cells is discussed in more detail in copending PCTPublished Patent Applications claiming priority to U.S. Ser. Nos.08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997respectively, the entire contents of which is hereby incorporated byreference. It is believed that these modified oligonucleotides may showmore stimulatory activity due to enhanced nuclease resistance, increasedcellular uptake, increased protein binding, and/or altered intracellularlocalization.

Both phosphorothioate and phosphodiester oligonucleotides containing CpGmotifs are active in immune cells. However, based on the concentrationneeded to induce CpG specific effects, the nuclease resistantphosphorothioate backbone CpG oligonucleotides are more potent (2 μg/mlfor the phosphorothioate vs. a total of 90 μg/ml for phosphodiester).

Other stabilized oligonucleotides include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Oligonucleotides which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

The nucleic acid sequences of the invention which are useful asadjuvants are those broadly described above and disclosed in PCTPublished Patent Applications claiming priority to U.S. Ser. Nos.08/738,652 and 08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997respectively. Exemplary sequences include but are not limited to thoseimmunostimulatory sequences shown in Table 1.

The stimulation index of a particular immunostimulatory CpG DNA can betested in various immune cell assays. Preferably, the stimulation indexof the CpG oligonucleotide with regard to B cell proliferation is atleast about 5, preferably at least about 10, more preferably at leastabout 15 and most preferably at least about 20 as determined byincorporation of ³H uridine in a murine B cell culture, which has beencontacted with 20 μM of oligonucleotide for 20 h at 37° C. and has beenpulsed with 1 μCi of ³H uridine; and harvested and counted 4 h later asdescribed in detail in copending PCT Published Patent Applicationsclaiming priority to U.S. Ser. Nos. 08/738,652 and 08/960,774, filed onOct. 30, 1996 and Oct. 30, 1997 respectively. For use in vivo, forexample, it is important that the CpG oligonucleotide and adjuvant becapable of effectively inducing activation of Ig expressing B cells.Oligonucleotides which can accomplish this include, for example, but arenot limited to those oligonucleotides described in PCT Published PatentApplications claiming priority to U.S. Ser. Nos. 08/738,652 and08/960,774, filed on Oct. 30, 1996 and Oct. 30, 1997 respectively.

The oligonucleotide containing at least one unmethylated CpG is used incombination with a non-nucleic acid adjuvant and an antigen to activatethe immune response. A “non-nucleic acid adjuvant” is any molecule orcompound except for the CpG oligonucleotides described herein which canstimulate the humoral and/or cellular immune response. Non-nucleic acidadjuvants include, for instance, adjuvants that create a depo effect,immune stimulating adjuvants, and adjuvants that create a depo effectand stimulate the immune system. In infants, the oligonucleotidecontaining at least one unmethylated CpG is used alone or in combinationwith a non-nucleic acid adjuvant and an antigen to activate a cellularimmune response.

An “adjuvant that creates a depo effect” as used herein is an adjuvantthat causes the antigen to be slowly released in the body, thusprolonging the exposure of immune cells to the antigen. This class ofadjuvants includes but is not limited to alum (e.g., aluminum hydroxide,aluminum phosphate); or emulsion-based formulations including mineraloil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion,oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants(e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (asqualene-in-water emulsion stabilized with Span 85 and Tween 80; ChironCorporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsioncontaining a stabilizing detergent and a micelle-forming agent; IDEC,Pharmaceuticals Corporation, San Diego, Calif.).

An “immune stimulating adjuvant” is an adjuvant that causes activationof a cell of the immune system. It may, for instance, cause an immunecell to produce and secrete cytokines. This class of adjuvants includesbut is not limited to saponins purified from the bark of the Q.saponaria tree, such as QS21 (a glycolipid that elutes in the 21^(st)peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA); derivatives of lipopolysaccharides suchas monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonylmuramyldipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related tolipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.).

“Adjuvants that create a depo effect and stimulate the immune system”are those compounds which have both of the above-identified functions.This class of adjuvants includes but is not limited to ISCOMS(Immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxpropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

When the CpG oligonucleotide containing at least one unmethylated CpG isadministered in conjunction with another adjuvant, the CpGoligonucleotide can be administered before, after, and/or simultaneouslywith the other adjuvant. For instance, the combination of adjuvants maybe administered with a priming dose of antigen. Either or both of theadjuvants may then be administered with the boost dose. Alternatively,the combination of adjuvants may be administered with a boost dose ofantigen. Either or both of the adjuvants may then be administered withthe prime dose. A “prime dose” is the first dose of antigen administeredto the subject. In the case of a subject that has an infection the primedose may be the initial exposure of the subject to the infectiousmicrobe and thus the combination of adjuvants is administered to thesubject with the boost dose. A “boost dose” is a second or third, etc.,dose of antigen administered to a subject that has already been exposedto the antigen. In some cases the prime dose administered with thecombination of adjuvants is so effective that a boost dose is notrequired to protect a subject at risk of infection from being infected.In cases where the combination of adjuvants is given without antigen,with repeated administrations, CpG oligonucleotides or one of thecomponents in the combination may be given alone for one or more of theadministrations.

The CpG oligonucleotide containing at least one unmethylated CpG canhave an additional efficacy (e.g., antisense) in addition to its abilityto enhance antigen-specific immune responses.

An “antigen” as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to peptides,polypeptides, cells, cell extracts, polysaccharides, polysaccharideconjugates, lipids, glycoliopids and carbohydrates. Antigens may begiven in a crude, purified or recombinant form and polypeptide/peptideantigens, including peptide mimics of polysaccharides, may also beencoded within nucleic. The term antigen broadly includes any type ofmolecule which is recognized by a host immune system as being foreign.Antigens include but are not limited to cancer antigens, microbialantigens, and allergens.

A “cancer antigen” as used herein is a compound, such as a peptide,associated with a tumor or cancer cell surface and which is capable ofprovoking an immune response when expressed on the surface of an antigenpresenting cell in the context of an MHC molecule. Cancer antigens canbe prepared from cancer cells either by preparing crude extracts ofcancer cells, for example, as described in Cohen, et al., 1994, CancerResearch, 54:1055, by partially purifying the antigens, by recombinanttechnology, or by de novo synthesis of known antigens. Cancer antigensinclude antigens that are recombinately an immunogenic portion of or awhole tumor or cancer. Such antigens can be isolated or preparedrecombinatly or by any other means known in the art.

A “microbial antigen” as used herein is an antigen of a microorganismand includes but is not limited to infectious virus, infectiousbacteria, infectious parasites and infectious fungi. Such antigensinclude the intact microorganism as well as natural isolates andfragments or derivatives thereof and also synthetic compounds which areidentical to or similar to natural microorganism antigens and induce animmune response specific for that microorganism. A compound is similarto a natural microorganism antigen if it induces an immune response(humoral and/or cellular) to a natural microorganism antigen. Most suchantigens are used routinely in the art and are well known to those ofordinary skill in the art. Another example is a peptide mimic of apolysaccharide antigen.

Examples of infectious virus that have been found in humans include butare not limited to: Retroviridae (e.g. human immunodeficiency viruses,such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, orHIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polioviruses, hepatitis A virus; enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses); Calciviridae (e.g. strains that causegastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubellaviruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellowfever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida , Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Examples of infectiousparasites include Plasmodium such as Plasmodium falciparum, Plasmodiummalariae, Plasmodium ovate, and Plasmodium vivax. Other infectiousorganisms (i.e. protists) include Toxoplasma gondii.

Other medically relevant microorganisms have been descried extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

Although many of the microbial antigens described above relate to humandisorders, the invention is also useful for treating other nonhumanvertebrates. Nonhuman vertebrates are also capable of developinginfections which can be prevented or treated with the synergisticcombination of adjuvants disclosed herein. For instance, in addition tothe treatment of infectious human diseases, the methods of the inventionare useful for treating infections of animals.

As used herein, the term “treat”, “treated”, or “treating” when usedwith respect to an infectious disease refers to a prophylactic treatmentwhich increases the resistance of a subject (a subject at risk ofinfection) to infection with a pathogen or, in other words, decreasesthe likelihood that the subject will become infected with the pathogen,as well as a treatment after the subject (a subject who has beeninfected) has become infected in order to fight the infection, e.g.,reduce or eliminate the infection or prevent it from becoming worse.Many vaccines for the treatment of non-human vertebrates are disclosedin Bennett, K. Compendium of Veterinary Products, 3rd ed. North AmericanCompendiums, Inc., 1995.

As discussed above, antigens include infectious microbes such as virus,bacteria, parasites and fungi and fragments thereof, derived fromnatural sources or synthetically. Infectious virus of both human andnon-human vertebrates, include retroviruses, RNA viruses and DNAviruses. This group of retroviruses includes both simple retrovirusesand complex retroviruses. The simple retroviruses include the subgroupsof B-type retroviruses, C-type retroviruses and D-type retroviruses. Anexample of a B-type retrovirus is mouse mammary tumor virus (MMTV). TheC-type retroviruses include subgroups C-type group A (including Roussarcoma virus (RSV), avian leukemia virus (ALV), and avianmyeloblastosis virus (AMV)) and C-type group B (including murineleukemia virus (MLV), feline leukemia virus (FeLV), murine sarcoma virus(MSV), gibbon ape leukemia virus (GALV), spleen necrosis virus (SNV),reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV)). TheD-type retroviruses include Mason-Pfizer monkey virus (MPMV) and simianretrovirus type 1 (SRV-1). The complex retroviruses include thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, Visnavirus, feline immunodeficiency virus (FIV), and equine infectious anemiavirus (EIAV). The T-cell leukemia viruses include HTLV-1, HTLV-II,simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV).The foamy viruses include human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV).

Examples of other RNA viruses that are antigens in vertebrate animalsinclude, but are not limited to, the following: members of the familyReoviridae, including the genus Orthoreovirus (multiple serotypes ofboth mammalian and avian retroviruses), the genus Orbivirus (Bluetonguevirus, Eugenangee virus, Kemerovo virus, African horse sickness virus,and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovineor ovine rotavirus, avian rotavirus); the family Picomaviridae,including the genus Enterovirus (poliovirus, Coxsackie virus A and B,enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus,Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirusmuris, Bovine enteroviruses, Porcine enteroviruses , the genusCardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genusRhinovirus (Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); forest virus, Sindbisvirus, Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,Venezuelan equine encephalitis virus, Western equine encephalitisvirus), the genus Flavirius (Mosquito borne yellow fever virus, Denguevirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley encephalitis virus, West Nile virus, Kunjin virus, CentralEuropean tick borne virus, Far Eastern tick borne virus, Kyasanur forestvirus, Louping III virus, Powassan virus, Omsk hemorrhagic fever virus),the genus Rubivirus (Rubella virus), the genus Pestivirus (Mucosaldisease virus, Hog cholera virus, Border disease virus); the familyBunyaviridae, including the genus Bunyvirus (Bunyamwera and relatedviruses, California encephalitis group viruses), the genus Phlebovirus(Sandfly fever Sicilian virus, Rift Valley fever virus), the genusNairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep diseasevirus), and the genus Uukuvirus (Uukuniemi and related viruses); thefamily Orthomyxoviridae, including the genus Influenza virus (Influenzavirus type A, many human subtypes); Swine influenza virus, and Avian andEquine Influenza viruses; influenza type B (many human subtypes), andinfluenza type C (possible separate genus); the family paramyxoviridae,including the genus Paramyxovirus (Parainfluenza virus type 1, Sendaivirus, Hemadsorption virus, Parainfluenza viruses types 2 to 5,Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (Measlesvirus, subacute sclerosing panencephalitis virus, distemper virus,Rinderpest virus), the genus Pneumovirus (respiratory syncytial virus(RSV), Bovine respiratory syncytial virus and Pneumonia virus of mice);the family Rhabdoviridae, including the genus Vesiculovirus (VSV),Chandipura virus, Flanders-Hart Park virus), the genus Lyssavirus(Rabies virus), fish Rhabdoviruses, and two probable Rhabdoviruses(Marburg virus and Ebola virus); the family Arenaviridae, includingLymphocytic choriomeningitis virus (LCM), Tacaribe virus complex, andLassa virus; the family Coronoaviridae, including Infectious BronchitisVirus (IBV), Mouse Hepatitis virus, Human enteric corona virus, andFeline infectious peritonitis (Feline coronavirus).

Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to: the family Poxviridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine, monkeys and rodents); thegamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus,Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, guinea pigherpes virus, Lucke tumor virus); the family Adenoviridae, including thegenus Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simianadenoviruses (at least 23 serotypes), infectious canine hepatitis, andadenoviruses of cattle, pigs, sheep, frogs and many other species, thegenus Aviadenovirus (Avian adenoviruses); and non-cultivatableadenoviruses; the family Papoviridae, including the genus Papillomavirus(Human papilloma viruses, bovine papilloma viruses, Shope rabbitpapilloma virus, and various pathogenic papilloma viruses of otherspecies), the genus Polyomavirus (polyomavirus, Simian vacuolating agent(SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus,and other primate polyoma viruses such as Lymphotrophic papillomavirus); the family Parvoviridae including the genus Adeno-associatedviruses, the genus Parvovirus (Feline panleukopenia virus, bovineparvovirus, canine parvovirus, Aleutian mink disease virus, etc).Finally, DNA viruses may include viruses which do not fit into the abovefamilies such as Kuru and Creutzfeldt-Jacob disease viruses and chronicinfectious neuropathic agents (CHINA virus).

Each of the foregoing lists is illustrative, and is not intended to belimiting.

In addition to the use of the combination of CpG oligonucleotides andnon-nucleic acid adjuvants to induce an antigen specific immune responsein humans, the methods of the preferred embodiments are particularlywell suited for treatment of birds such as hens, chickens, turkeys,ducks, geese, quail, and pheasant. Birds are prime targets for manytypes of infections.

Hatching birds are exposed to pathogenic microorganisms shortly afterbirth. Although these birds are initially protected against pathogens bymaternal derived antibodies, this protection is only temporary, and thebird's own immature immune system must begin to protect the bird againstthe pathogens. It is often desirable to prevent infection in young birdswhen they are most susceptible. It is also desirable to prevent againstinfection in older birds, especially when the birds are housed in closedquarters, leading to the rapid spread of disease. Thus, it is desirableto administer the CpG oligonucleotide and the non-nucleic acid adjuvantof the invention to birds to enhance an antigen-specific immune responsewhen antigen is present. The CpG oligonucleotide and the non-nucleicacid adjuvant of the invention could also be administered to birdswithout antigen to protect against infection of a wide variety ofpathogens.

An example of a common infection in chickens is chicken infectiousanemia virus (CIAV). CIAV was first isolated in Japan in 1979 during aninvestigation of a Marek's disease vaccination break (Yuasa et al.,1979, Avian Dis. 23:366-385). Since that time, CIAV has been detected incommercial poultry in all major poultry producing countries (van Bulowet al., 1991, pp. 690-699) in Diseases of Poultry, 9th edition, IowaState University Press).

CIAV infection results in a clinical disease, characterized by anemia,hemorrhage and immunosuppression, in young susceptible chickens. Atrophyof the thymus and of the bone marrow and consistent lesions ofCIAV-infected chickens are also characteristic of CIAV infection.Lymphocyte depletion in the thymus, and occasionally in the bursa ofFabricius, results in immunosuppression and increased susceptibility tosecondary viral, bacterial, or fungal infections which then complicatethe course of the disease. The immunosuppression may cause aggravateddisease after infection with one or more of Marek's disease virus (MDV),infectious bursal disease virus, reticuloendotheliosis virus,adenovirus, or reovirus. It has been reported that pathogenesis of MDVis enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it hasbeen reported that CIAV aggravates the signs of infectious bursaldisease (Rosenberger et al., 1989, Avian Dis. 33:707-713). Chickensdevelop an age resistance to experimentally induced disease due to CAA.This is essentially complete by the age of 2 weeks, but older birds arestill susceptible to infection (Yuasa, N. et al., 1979 supra; Yuasa, N.et al., Arian Diseases 24, 202-209, 1980). However, if chickens aredually infected with CAA and an immunosuppressive agent (IBDV, MDV etc.)age resistance against the disease is delayed (Yuasa, N. et al., 1979and 1980 supra; Bulow von V. et al., J. Veterinary Medicine 33, 93-116,1986). Characteristics of CIAV that may potentiate disease transmissioninclude high resistance to environmental inactivation and some commondisinfectants. The economic impact of CIAV infection on the poultryindustry is clear from the fact that 10% to 30% of infected birds indisease outbreaks die.

Vaccination of birds, like other vertebrate animals can be performed atany age. Normally, vaccinations are performed at up to 12 weeks of agefor a live microorganism and between 14-18 weeks for an inactivatedmicroorganism or other type of vaccine. For in ovo vaccination,vaccination can be performed in the last quarter of embryo development.The vaccine may be administered subcutaneously, by spray, orally,intraocularly, intratracheally, nasally, in ovo or by other methodsdescribed herein. Thus, the CpG oligonucleotide and non-nucleic acidadjuvant of the invention can be administered to birds and othernon-human vertebrates using routine vaccination schedules and theantigen is administered after an appropriate time period as describedherein.

Cattle and livestock are also susceptible to infection. Disease whichaffect these animals can produce severe economic losses, especiallyamongst cattle. The methods of the invention can be used to protectagainst infection in livestock, such as cows, horses, pigs, sheep, andgoats. The CpG oligonucleotide and the non-nucleic acid adjuvant of theinvention could also be administered with antigen for antigen-specificprotection of long duration or without antigen for short term protectionagainst a wide variety of diseases, including shipping fever.

Cows can be infected by bovine viruses. Bovine viral diarrhea virus(BVDV) is a small enveloped positive-stranded RNA virus and isclassified, along with hog cholera virus (HOCV) and sheep border diseasevirus (BDV), in the pestivirus genus. Although, Pestiviruses werepreviously classified in the Togaviridae family, some studies havesuggested their reclassification within the Flaviviridae family alongwith the flavivirus and hepatitis C virus (HCV) groups (Francki, et al.,1991).

BVDV, which is an important pathogen of cattle can be distinguished,based on cell culture analysis, into cytopathogenic (CP) andnoncytopathogenic (NCP) biotypes. The NCP biotype is more widespreadalthough both biotypes can be found in cattle. If a pregnant cow becomesinfected with an NCP strain, the cow can give birth to a persistentlyinfected and specifically immunotolerant calf that will spread virusduring its lifetime. The persistently infected cattle can succumb tomucosal disease and both biotypes can then be isolated from the animal.Clinical manifestations can include abortion, teratogenesis, andrespiratory problems, mucosal disease and mild diarrhea. In addition,severe thrombocytopenia, associated with herd epidemics, that may resultin the death of the animal has been described and strains associatedwith this disease seem more virulent than the classical BVDVs.

Equine herpesviruses (EHV) comprise a group of antigenically distinctbiological agents which cause a variety of infections in horses rangingfrom subclinical to fatal disease. These include Equine herpesvirus-1(EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated withepidemics of abortion, respiratory tract disease, and central nervoussystem disorders. Primary infection of upper respiratory tract of younghorses results in a febrile illness which lasts for 8 to 10 days.Immunologically experienced mares may be reinfected via the respiratorytract without disease becoming apparent, so that abortion usually occurswithout warning. The neurological syndrome is associated withrespiratory disease or abortion and can affect animals of either sex atany age, leading to incoordination, weakness and posterior paralysis(Telford, E. A. R. et al., Virology 189, 304-316, 1992). Other EHV'sinclude EHV-2, or equine cytomegalovirus, EHV-3, equine coital exanthemavirus, and EHV-4, previously classified as EHV-1 subtype 2.

Sheep and goats can be infected by a variety of dangerous microorganismsincluding visna-maedi.

Primates such as monkeys, apes and macaques can be infected by simianimmunodeficiency virus. Inactivated cell-virus and cell-free wholesimian immunodeficiency vaccines have been reported to afford protectionin macaques (Stott et al. (1990) Lancet 36:1538-1541; Desrosiers et al.PNAS USA (1989) 86:6353-6357; Murphey-Corb et al. (1989) Science246:1293-1297; and Carlson et al. (1990) AIDS Res. Human Retroviruses6:1239-1246). A recombinant HIV gpl120vaccine has been reported toafford protection in chimpanzees (Berman et al. (1990) Nature345:622-625).

Cats, both domestic and wild, are susceptible to infection with avariety of microorganisms. For instance, feline infectious peritonitisis a disease which occurs in both domestic and wild cats, such as lions,leopards, cheetahs, and jaguars. When it is desirable to preventinfection with this and other types of pathogenic organisms in cats, themethods of the invention can be used to vaccinate cats to prevent themagainst infection.

Domestic cats may become infected with several retroviruses, includingbut not limited to feline leukemia virus (FeLV), feline sarcoma virus(FeSV), endogenous type C oncomavirus (RD-114), and felinesyncytia-forming virus (FeSFV). Of these, FeLV is the most significantpathogen, causing diverse symptoms, including lymphoreticular andmyeloid neoplasms, anemias, immune mediated disorders, and animmunodeficiency syndrome which is similar to human acquired immunedeficiency syndrome (AIDS). Recently, a particular replication-defectiveFeLV mutant, designated FeLV-AIDS, has been more particularly associatedwith immunosuppressive properties.

The discovery of feline T-lymphotropic lentivirus (also referred to asfeline immunodeficiency) was first reported in Pedersen et al. (1987)Science 235:790-793. Characteristics of FIV have been reported inYamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S;Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al.(1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV havebeen reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA86:2448-2452 and 86:4355-4360.

Feline infectious peritonitis (FIP) is a sporadic disease occurringunpredictably in domestic and wild Felidae. While FIP is primarily adisease of domestic cats, it has been diagnosed in lions, mountainlions, leopards, cheetahs, and the jaguar. Smaller wild cats that havebeen afflicted with FIP include the lynx and caracal, sand cat, andpallas cat. In domestic cats, the disease occurs predominantly in younganimals, although cats of all ages are susceptible. A peak incidenceoccurs between 6 and 12 months of age. A decline in incidence is notedfrom 5 to 13 years of age, followed by an increased incidence in cats 14to 15 years old.

Viral, bacterial and parasitic diseases in fin-fish, shellfish or otheraquatic life forms pose a serious problem for the aquaculture industry.Owing to the high density of animals in the hatchery tanks or enclosedmarine farming areas, infectious diseases may eradicate a largeproportion of the stock in, for example, a fin-fish, shellfish, or otheraquatic life forms facility. Prevention of disease is a more desiredremedy to these threats to fish than intervention once the disease is inprogress. Vaccination of fish is the only preventative method which mayoffer long-term protection through immunity. Fish are currentlyprotected against a variety of bacterial infections with whole killedvaccines with oli adjuvants, but there is only one licensed vaccine forfish against a viral disease. Nucleic acid based vaccinations aredescribed in U.S. Pat. No. 5,780,448 issued to Davis and these have beenshown to be protective against at least two different viral diseases.

The fish immune system has many features similar to the mammalian immunesystem, such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Vaccines can be administered orally or by immersion orinjection.

Aquaculture species include but are not limited to fin-fish, shellfish,and other aquatic animals. Fin-fish include all vertebrate fish, whichmay be bony or cartilaginous fish, such as, for example, salmonids,carp, catfish, yellowtail, seabream, and seabass. Salmonids are a familyof fin-fish which include trout (including rainbow trout), salmon, andArctic char. Examples of shellfish include, but are not limited to,clams, lobster, shrimp, crab, and oysters. Other cultured aquaticanimals include, but are not limited to eels, squid, and octopi.

Polypeptides of viral aquaculture pathogens include but are not limitedto glycoprotein (G) or nucleoprotein (N) of viral hemorrhagic septicemiavirus (VHSV); G or N proteins of infectious hematopoietic necrosis virus(IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreaticnecrosis virus (IPNV); G protein of spring viremia of carp (SVC); and amembrane-associated protein, tegumin or capsid protein or glycoproteinof channel catfish virus (CCV).

Polypeptides of bacterial pathogens include but are not limited to aniron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (IROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein,aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

Polypeptides of a parasitic pathogen include but are not limited to thesurface antigens of Ichthyophthirius.

An “allergen” refers to a substance (antigen) that can induce anallergic or asthmatic response in a susceptible subject. The list ofallergens is enormous and can include pollens, insect venoms, animaldander dust, fungal spores and drugs (e.g. penicillin). Examples ofnatural, animal and plant allergens include but are not limited toproteins specific to the following genuses: Canine (Canis familiaris);Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felisdomesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Loliumperenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica);Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa);Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata);Parietaria (e.g. Parietaria officinalis or Parietaria judaica);Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum);Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica andCupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperusvirginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuyaorientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta(e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale(e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g.Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poapratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g.Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum);Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostisalba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

In some aspects of the invention the antigen is a polypeptide. Minormodifications of the primary amino acid sequences of polypeptideantigens may also result in a polypeptide which has substantiallyequivalent antigenic activity as compared to the unmodified counterpartpolypeptide. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein as long as antigenicity stillexists. The polypeptide may be, for example, a viral polypeptide. Onenon-limiting example of an antigen useful according to the invention isthe hepatitis B surface antigen. Experiments using this antigen aredescribed in the Examples below.

The term “substantially purified” as used herein refers to a polypeptidewhich is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify viral or bacterial polypeptides using standardtechniques for protein purification. The substantially pure polypeptidewill often yield a single major band on a non-reducing polyacrylamidegel. In the case of partially glycosylated polypeptides or those thathave several start codons, there may be several bands on a non-reducingpolyacrylamide gel, but these will form a distinctive pattern for thatpolypeptide. The purity of the viral or bacterial polypeptide can alsobe determined by amino-terminal amino acid sequence analysis.

The invention also utilizes polynucleotides encoding the antigenicpolypeptides. It is envisioned that the antigen may be delivered to thesubject in a nucleic acid molecule which encodes for the antigen suchthat the antigen must be expressed in vivo. The nucleic acid encodingthe antigen is operatively linked to a gene expression sequence whichdirects the expression of the antigen nucleic acid within a eukaryoticcell. The “gene expression sequence” is any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient transcription and translation of theantigen nucleic acid to which it is operatively linked. The geneexpression sequence may, for example, be a mammalian or viral promoter,such as a constitutive or inducible promoter. Constitutive mammalianpromoters include, but are not limited to, the promoters for thefollowing genes: hypoxanthine phosphoribosyl transferase (HPTR),adenosine deaminase, pyruvate kinase, $-actin promoter, muscle creatinekinase promoter, human elongation factor promoter and other constitutivepromoters. Exemplary viral promoters which function constitutively ineukaryotic cells include, for example, promoters from the simian virus(e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus(HIV), rous sarcoma virus, cytomegalovirus (CMV), Rous sarcoma virus(RSV), hepatitis B virus (HBV), the long terminal repeats (LTR) ofMoloney leukemia virus and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined antigen nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

The antigen nucleic acid is operatively linked to the gene expressionsequence. As used herein, the antigen nucleic acid sequence and the geneexpression sequence are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the antigen coding sequence underthe influence or control of the gene expression sequence. Two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ gene expression sequence results in the transcription of theantigen sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the antigen sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to anantigen nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that antigen nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

The antigen nucleic acid sequence may encode a protein, polypeptide,peptide, or peptide mimic of a polysaccharide. It may also encode morethan one antigenic component as a fusion construct. More than oneantigen-encoding sequence may be included in the same plasmid vector andthese may be linked to the same or different gene expression sequences.

The antigen nucleic acid of the invention may be delivered to the immunesystem alone or in association with a vector. In its broadest sense, a“vector” is any vehicle capable of facilitating the transfer of theantigen nucleic acid to the cells of the immune system and preferablyAPCs so that the antigen can be expressed and presented on the surfaceof an APC. Preferably, the vector transports the nucleic acid to theimmune cells with reduced degradation relative to the extent ofdegradation that would result in the absence of the vector. The vectoroptionally includes the above-described gene expression sequence toenhance expression of the antigen nucleic acid in APCs. In general, thevectors useful in the invention include, but are not limited to,plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the antigen nucleic acid sequences. Viral vectors are apreferred type of vector and include, but are not limited to nucleicacid sequences from the following viruses: retrovirus, such as moloneymurine leukemia virus, harvey murine sarcoma virus, murine mammary tumorvirus, and rouse sarcoma virus; adenovirus, adeno-associated virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual”, W. H.Freeman C.O., New York (1990) and Murry, E. J. Ed. “Methods in MolecularBiology”, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

Preferred virus for certain applications are the adeno-virus andadeno-associated virus which are double-stranded DNA viruses that havealready been approved for human use in gene therapy and immunotherapytrials. The adeno-associated virus can be engineered to bereplication-deficient and is capable of infecting a wide range of celltypes and species. It further has advantages such as, heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hemopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See e.g., Sanbrook et al., “Molecular Cloning: A LaboratoryManual”, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, andpBlueScript. Other plasmids are well-known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids such as those used for DNA vaccines may be delivered by avariety of parenteral, mucosal and topical routes. For example theplasmid DNA can be injected by intramuscular, intradermal, subcutaneousor other routes. It may also be administered by intranasal sprays ordrops, rectal suppository and orally. It may also be administered intothe epidermis or a mucosal surface using a gene-gun. The plasmids may begiven in an aqueous solution, dried onto gold particles or inassociation with another DNA delivery system including but not limitedto liposomes, dendrimers, cochleate and microencapsulation.

It has recently been discovered that gene carrying plasmids can bedelivered to the immune system using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to immune cells, e.g. dendritic cells,probably by passing through the gut barrier. High levels of immuneprotection have been established using this methodology.

In other aspects the invention includes a method for immunizing aninfant by administering to an infant an antigen and an oligonucleotidecontaining at least one unmethylated CpG dinucleotide in an effectiveamount for inducing cell mediated immunity in the infant. In someembodiments the infant is also administered at least one non-nucleicacid adjuvant, as described above. Cell mediated immunity, as usedherein, refers to an immune response which involves an antigen specificT cell reaction. The presence of cell mediated immunity can bedetermined directly by the induction of Th1 cytokines (e.g., IFN-γ,IL-12) and antigen-specific cytotoxic T-cell lymphocytes (CTL). Thepresence of cell mediated immunity is also indicated indirectly by theisotype of antigen-specific antibodies that are induced (e.g.IgG2a>>IgG1 in mice). Thus, if Th1 cytokines or CTL or TH-likeantibodies are induced, cell mediated immunity is induced according tothe invention. As discussed above, Th1 cytokines include but are notlimited to IL-12 and IFN-γ.

Neonates (newborn) and infants (which include humans three months of ageand referred to hereinafter as infants) born in HBV endemic areasrequire particularly rapid induction of strong HBV-specific immunityowing to the high rate of chronicity resulting from infection at a youngage. Without immunoprophylaxis, 70-90% of infants born to motherspositive for both HBsAg and the “e” antigen (HBeAg) become infected andalmost all of these become chronic carriers (Stevens et al. , 1987).Even when vaccinated with a four dose regime of the HBV subunit vaccinecommencing on the day of birth, 20% of such infants became chronicallyinfected and this was reduced to only 15% if they were also givenHBV-specific immunoglobulin (Chen et al , 1996). HBV chronicity resultsin 10-15% of individuals infected as adolescents or adults, but 90-95%for those infected (either vertically or horizontally) as infants. CpGoligonucleotides may be used, according to the invention, to reduce thisfurther owing to a more rapid appearance and higher titers of anti-HBsantibodies and the induction of HBV-specific CTL, which could help clearvirus from the liver of babies infected in utero, and which likelyaccount for most of the failures with infant vaccination.

The invention further provides a method of modulating the level of acytokine. The term “modulate” envisions the suppression of expression ofa particular cytokine when lower levels are desired, or augmentation ofthe expression of a particular cytokine when higher levels are desired.Modulation of a particular cytokine can occur locally or systemically.CpG oligonucleotides can directly activate macrophages and dendriticcells to secrete cytokines. No direct activation of proliferation orcytokine secretion by highly purified T cells has been found, althoughthey are induced to secrete cytokines by cytokines secreted frommacrophages and may be costimulated through the T cell Receptor.Cytokine profiles determine T cell regulatory and effector functions inimmune responses. In general, Th1-type cytokines are induced, thus theimmunostimulatory nucleic acids promote a Th1 type antigen-specificimmune response including cytotoxic T-cells.

Cytokines also play a role in directing the T cell response. Helper(CD4⁺) T cells orchestrate the immune response of mammals throughproduction of soluble factors that act on other immune system cells,including B and other T cells. Most mature CD4⁺ T helper cells expressone of two cytokine profiles: Th1 or Th2. Th1 cells secrete IL-2, IL-3,IFN-γ, GM-CSF and high levels of TNF-α. Th2 cells express IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF-α. The Th1subset promotes both cell-mediated immunity, and humoral immunity thatis characterized by immunoglobulin class switching to IgG_(2a) in mice.Th1 responses may also be associated with delayed-type hypersensitivityand autoimmune disease. The Th2 subset induces primarily humoralimmunity and induce class switching to IgG₁ and IgE. The antibodyisotypes associated with Th1 responses generally have good neutralizingand opsonizing capabilities whereas those associated with Th2 responsesare associated more with allergic responses.

Several factors have been shown to influence commitment to Th1 or Th2profiles. The best characterized regulators are cytokines. IL-12 andIFN-γ are positive Th1 and negative Th2 regulators. IL-12 promotes IFN-γproduction, and IFN-γ provides positive feedback for IL-12. IL-4 andIL-10 appear to be required for the establishment of the Th2 cytokineprofile and to down-regulate Th1 cytokine production; the effects ofIL-4 are in some cases dominant over those of IL-12. IL-13 was shown toinhibit expression of inflammatory cytokines, including IL-12 and TNF-αby LPS-induced monocytes, in a way similar to IL-4. The IL-12 p40homodimer binds to the IL-12 receptor and may antagonizes IL-12biological activity; thus it blocks the pro-Th1 effects of IL-12 in someanimals.

In other aspects the invention includes a method of inducing a Th1immune response in a subject by administering to the subject acombination of adjuvants in an effective amount for inducing a Th1immune response. The combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant. It was not previously knownthat when CpG was combined with a non-nucleic acid adjuvant, asdescribed above, that the combination would produce an immune responsewith a Th1 profile to an extent that the individual adjuvants could notproduce alone. Preferably the extent of the Th profile produced by thecombination of adjuvants is synergistic. Another aspect of the inventionis to induce a Th response by using CPG with a non-nucleic acid adjuvantthat by itself induces a Th2 response.

As described above a Th2 profile is characterized by production of IL-4and IL-10. Non-nucleic acid adjuvants that induce Th2 or weak Th1responses include but are not limited to alum, saponins, oil-in-waterand other emulsion formulations and SB-As4. Adjuvants that induce Th1responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN-γ,and SB-AS2. When the CpG oligonucleotide is administered with anon-nucleic acid adjuvant the combination of adjuvants causes acommitment to a Th1 profile, that neither the adjuvant nor the CpGoligonucleotide is capable of producing on its own. Furthermore, if thenon-nucleic acid adjuvant on its own induces a Th2 response, theaddition of CpG oligonucleotide can overcome this Th2 bias and induce aTh1 response that may be even more Th1-like than with CpG alone.

The combination of adjuvants may be administered simultaneously orsequentially. When the adjuvants are administered simultaneously theycan be administered in the same or separate formulations, and in thelatter case at the same or separate sites, but are administered at thesame time. The adjuvants are administered sequentially, when theadministration of the at least two adjuvants is temporally separated.The separation in time between the administration of the two adjuvantsmay be a matter of minutes or it may be longer. The separation in timeis less than 14 days, and more preferably less than 7 days, and mostpreferably less than 1 day. The separation in time may also be with oneadjuvant at prime and one at boost, or one at prime and the combinationat boost, or the combination at prime and one at boost.

For use in the instant invention, the nucleic acids can be synthesizedde novo using any of a number of procedures well known in the art. Forexample, the b-cyanoethyl phosphoramidite method (Beaucage, S. L., andCaruthers, M. H., Tet. Let. 22:1859, 1981); nucleoside H-phosphonatemethod (Garegg et al., Tet. Let. 27:4051-4054, 1986; Froehler et al.,Nucl. Acid. Res. 14:5399-5407, 1986,; Garegg et al., Tet. Let.27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622, 1988). Thesechemistries can be performed by a variety of automated oligonucleotidesynthesizers available in the market. Alternatively, CpG dinucleotidescan be produced on a large scale in plasmids, (see Sambrook, T., et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratoryPress, New York, 1989) which after being administered to a subject aredegraded into oligonucleotides. Such plasmids may also encode othergenes to be expressed such as an antigen-encoding gene in the case of aDNA vaccine. Oligonucleotides can be prepared from existing nucleic acidsequences (e.g., genomic or cDNA) using known techniques, such as thoseemploying restriction enzymes, exonucleases or endonucleases.

For use in vivo, nucleic acids are preferably relatively resistant todegradation (e.g., via endo-and exo-nucleases). Secondary structures,such as stem loops, can stabilize nucleic acids against degradation.Alternatively, nucleic acid stabilization can be accomplished viaphosphate backbone modifications. A preferred stabilized nucleic acidhas at least a partial phosphorothioate modified backbone.Phosphorothioates may be synthesized using automated techniquesemploying either phosphoramidate or H-phosphonate chemistries. Aryl-andalkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.4,469,863; and alkylphosphotriesters (in which the charged oxygen moietyis alkylated as described in U.S. Pat. No. 5,023,243 and European PatentNo. 092,574) can be prepared by automated solid phase synthesis usingcommercially available reagents. Methods for making other DNA backbonemodifications and substitutions have been described (Uhlmann, E. andPeyman, A., Chem. Rev. 90:544, 1990; Goodchild, J., Bioconjugate Chem.1:165, 1990).

For administration in vivo, nucleic acids may be associated with amolecule that results in higher affinity binding to target cell (e.g.,B-cell, monocytic cell and natural killer (NK) cell) surfaces and/orincreased cellular uptake by target cells to form a “nucleic aciddelivery complex.” Nucleic acids can be ionically or covalentlyassociated with appropriate molecules using techniques which are wellknown in the art. A variety of coupling or cross-linking agents can beused, e.g., protein A, carbodiimide, andN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Nucleic acids canalternatively be encapsulated in liposomes or virosomes using well-knowntechniques.

Nucleic acids containing an appropriate unmethylated CpG can beeffective in any mammal, preferably a human. Different nucleic acidscontaining an umnethylated CpG can cause optimal immune stimulationdepending on the mammalian species. Thus an oligonucleotide causingoptimal stimulation in humans may not cause optimal stimulation in amouse and vice versa. One of skill in the art can identify the optimaloligonucleotides useful for a particular mammalian species of interestusing routine assays described herein and/or known in the art.

The CpG ODN of the invention stimulate cytokine production (e.g., IL-6,IL-12, IFN-γ, TNF-α and GM-CSF) and B-cell proliferation in PBMC's takenfrom a subject such as a human. Specific, but nonlimiting examples ofsuch sequences include those presented in Table 1 below:

TABLE 1 sequences GCTAGACGTTAGCGT; (SEQ ID NO:1) GCTAGATGTTAGCGT; (SEQID NO:2) GCTAGACGTTAGCGT; (SEQ ID NO:3) GCTAGACGTTAGCGT; (SEQ ID NO:4)GCATGACGTTGAGCT; (SEQ ID NO:5) ATGGAAGGTCCAGCGTTCTC; (SEQ ID NO:6)ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:7) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:8)ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:9) ATGGAAGGTCCAACGTTCTC; (SEQ ID NO:10)GAGAACGCTGGACCTTCCAT; (SEQ ID NO:11) GAGAACGCTCGACCTTCCAT; (SEQ IDNO:12) GAGAACGCTCGACCTTCGAT; (SEQ ID NO:13) GAGAACGCTGGACCTTCCAT; (SEQID NO:14) GAGAACGATGGACCTTCCAT; (SEQ ID NO:15) GAGAACGCTCCAGCACTGAT;(SEQ ID NO:16) TCCATGTCGGTCCTGATGCT; (SEQ ID NO:17)TCCATGTCGGTCCTGATGCT; (SEQ ID NO:18) TCCATGACGTTCCTGATGCT; (SEQ IDNO:19) TCCATGTCGGTCCTGCTGAT; (SEQ ID NO:20) TCAACGTT; (SEQ ID NO:21)TCAGCGCT; (SEQ ID NO:22) TCATCGAT; (SEQ ID NO:23) TCTTCGAA; (SEQ IDNO:24) CAACGTT; (SEQ ID NO:25) CCAACGTT; (SEQ ID NO:26) AACGTTCT; (SEQID NO:27) TCAACGTC; (SEQ ID NO:28) ATGGACTCTCCAGCGTTCTC; (SEQ ID NO:29)ATGGAAGGTCCAACGTTCTC; (SEQ ID NO:30) ATCGACTCTCGAGCGTTCTC; (SEQ IDNO:31) ATGGAGGCTCCATCGTTCTC; (SEQ ID NO:32) ATCGACTCTCGAGCGTTCTC; (SEQID NO:33) ATCGACTCTCGAGCGTTCTC; (SEQ ID NO:34) TCCATGTCGGTCCTGATGCT;(SEQ ID NO:35) TCCATGCCGGTCCTGATGCT; (SEQ ID NO:36)TCCATGGCGGTCCTGATGCT; (SEQ ID NO:37) TCCATGACGGTCCTGATGCT; (SEQ IDNO:38) TCCATGTCGATCCTGATGCT; (SEQ ID NO:39) TCCATGTCGCTCCTGATGCT; (SEQID NO:40) TCCATGTCGTCCCTGATGCT; (SEQ ID NO:41) TCCATGACGTGCCTGATGCT;(SEQ ID NO:42) TCCATAACGTTCCTGATGCT; (SEQ ID NO:43)TCCATGACGTCCCTGATGCT; (SEQ ID NO:44) TCCATCACGTGCCTGATGCT; (SEQ IDNO:45) GGGGTCAACGTTGACGGGG; (SEQ ID NO:46) GGGGTCAGTCGTGACGGGG; (SEQ IDNO:47) GCTAGACGTTAGTGT; (SEQ ID NO:48) TCCATGTCGTTCCTGATGCT; (SEQ IDNO:49) ACCATGGACGATCTGTTTCCCCTC; (SEQ ID NO:50) TCTCCCAGCGTGCGCCAT; (SEQID NO:51) ACCATGGACGAACTGTTTCCCCTC; (SEQ ID NO:52)ACCATGGACGAGCTGTTTCCCCTC; (SEQ ID NO:53) ACCATGGACGACCTGTTTCCCCTC; (SEQID NO:54) ACCATGGACGTACTGTTTCCCCTC; (SEQ ID NO:55)ACCATGGACGGTCTGTTTCCCCTC; (SEQ ID NO:56) ACCATGGACGTTCTGTTTCCCCTC; (SEQID NO:57) CACGTTGAGGGGCAT; (SEQ ID NO:58) TCAGCGTGCGCC; (SEQ ID NO:59)ATGACGTTCCTGACGTT; (SEQ ID NO:60) TCTCCCAGCGGGCGCAT; (SEQ ID NO:61)TCCATGTCGTTCCTGTCGTT; (SEQ ID NO:62) TCCATAGCGTTCCTAGCGTT; (SEQ IDNO:63) TCGTCGCTGTCTCCCCTTCTT; (SEQ ID NO:64) TCCTGACGTTCCTGACGTT; (SEQID NO:65) TCCTGTCGTTCCTGTCGTT; (SEQ ID NO:66) TCCATGTCGTTTTTGTCGTT; (SEQID NO:67) TCCTGTCGTTCCTTGTCGTT; (SEQ ID NO:68) TCCTTGTCGTTCCTGTCGTT;(SEQ ID NO:69) TCCTGTCGTTTTTTGTCGTT; (SEQ ID NO:70)TCGTCGCTGTCTGCCCTTCTT; (SEQ ID NO:71) TCGTCGCTGTTGTCGTTTCTT; (SEQ IDNO:72) TCCATGCGTGCGTGCGTTTT; (SEQ ID NO:73) TCCATGCGTTGCGTTGCGTT; (SEQID NO:74) TCCACGACGTTTTCGACGTT; (SEQ ID NO:75) TCGTCGTTGTCGTTGTCGTT;(SEQ ID NO:76) TCGTCGTTTTGTCGTTTTGTCGTT; (SEQ ID NO:77)TCGTCGTTGTCGTTTTGTCGTT; (SEQ ID NO:78) GCGTGCGTTGTCGTTGTCGTT; (SEQ IDNO:79) TGTCGTTTGTCGTTTGTCGTT; (SEQ ID NO:80) TGTCGTTGTCGTTGTCGTTGTCGTT;(SEQ ID NO:81) TGTCGTTGTCGTTGTCGTT; (SEQ ID NO:82) TCGTCGTCGTCGTT; (SEQID NO:83) TGTCGTTGTCGTT; (SEQ ID NO:84) TCCATAGCGTTCCTAGCGTT; (SEQ IDNO:85) TCCATGACGTTCCTGACGTT; (SEQ ID NO:86) GTCGYT; (SEQ ID NO:87)TGTCGYT; (SEQ ID NO:88) AGCTATGACGTTCCAAGG; (SEQ ID NO:89)TCCATGACGTTCCTGACGTT; (SEQ ID NO:90) ATCGACTCTCGAACGTTCTC; (SEQ IDNO:91) TCCATGTCGGTCCTGACGCA; (SEQ ID NO:92) TCTTCGAT; (SEQ ID NO:93)ATAGGAGGTCCAACGTTCTC; (SEQ ID NO:94) GTCGTT (SEQ ID NO:95) GTCGTC (SEQID NO:96) TGTCGTT (SEQ ID NO:97) TGTCGCT (SEQ ID NO:98)

Preferred CpG ODN can effect at least about 500 pg/ml of TNF-α, 15 pg/mlIFN-γ, 70 pg/ml of GM-CSF 275 pg/ml of IL-6, 200 pg/ml IL-12, dependingon the therapeutic indication. These cytokines can be measured by assayswell known in the art. The oligonucleotides listed above or otherpreferred CpG ODN can effect at least about 10%, more preferably atleast about 15% and most preferably at least about 20% YAC-1 cellspecific lysis or at least about 30%, more preferably at least about35%, and most preferably at least about 40% 2C11 cell specific lysis, inassays well known in the art.

The term “effective amount” of a CpG oligonucleotide refers to theamount necessary or sufficient to realize a desired biologic effect. Forexample, an effective amount of an oligonucleotide containing at leastone unmethylated CpG and a non-nucleic acid adjuvant for treating aninfectious disorder is that amount necessary to cause the development ofan antigen specific immune response upon exposure to the microbe, thuscausing a reduction in the amount of microbe within the subject andpreferably to the eradication of the microbe. The effective amount forany particular application can vary depending on such factors as thedisease or condition being treated, the particular CpG oligonucleotidebeing administered (e.g. the number of unmethylated CpG motifs or theirlocation in the nucleic acid), the size of the subject, or the severityof the disease or condition. One of ordinary skill in the art canempirically determine the effective amount of a particular adjuvant andantigen without necessitating undue experimentation.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the adjuvant combination canbe administered to a subject by any mode allowing the oligonucleotide tobe taken up by the appropriate target cells. “Administering” thepharmaceutical composition of the present invention may be accomplishedby any means known to the skilled artisan. Preferred routes ofadministration include but are not limited to oral, transdermal (e.g.via a patch), parenteral injection (subcutaneous, intradermal,intravenous, parenteral, intraperitoneal, intrathecal, etc.), or mucosalintranasal, intratracheal, inhalation, and intrarectal, intravaginaletc). An injection may be in a bolus or a continuous infusion.

For example the pharmaceutical compositions according to the inventionare often administered by intramuscular or intradermal injection, orother parenteral means, or by biolistic “gene-gun” application to theepidermis. They may also be administered by intranasal application,inhalation, topically, intravenously, orally, or as implants, and evenrectal or vaginal use is possible. Suitable liquid or solidpharmaceutical preparation forms are, for example, aqueous or salinesolutions for injection or inhalation, microencapsulated, encochleated,coated onto microscopic gold particles, contained in liposomes,nebulized, aerosols, pellets for implantation into the skin, or driedonto a sharp object to be scratched into the skin. The pharmaceuticalcompositions also include granules, powders, tablets, coated tablets,(micro)capsules, suppositories, syrups, emulsions, suspensions, creams,drops or preparations with protracted release of active compounds, inwhose preparation excipients and additives and/or auxiliaries such asdisintegrants, binders, coating agents, swelling agents, lubricants,flavorings, sweeteners or solubilizers are customarily used as describedabove. The pharmaceutical compositions are suitable for use in a varietyof drug delivery systems. For a brief review of present methods for drugdelivery, see Langer, Science 249:1527-1533, 1990, which is incorporatedherein by reference.

The pharmaceutical compositions are preferably prepared and administeredin dose units. Liquid dose units are vials or ampoules for injection orother parenteral administration. Solid dose units are tablets, capsulesand suppositories. For treatment of a patient, depending on activity ofthe compound, manner of administration, purpose of the immunization(i.e., prophylactic or therapeutic), nature and severity of thedisorder, age and body weight of the patient, different doses may benecessary. The administration of a given dose can be carried out both bysingle administration in the form of an individual dose unit or elseseveral smaller dose units. Multiple administration of doses at specificintervals of weeks or months apart is usual for boosting theantigen-specific responses.

The adjuvants and antigens may be administered per se (neat) or in theform of a pharmaceutically acceptable salt. When used in medicine thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: hydrochloric, hydrobromic,sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulphonic, tartaric, citric, methane sulphonic, formic, malonic,succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the invention contain an effectiveamount of a combination of adjuvants and antigens optionally included ina pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid filler, dilutants or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being comingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

Compositions suitable for parenteral administration convenientlycomprise sterile aqueous preparations, which can be isotonic with theblood of the recipient. Among the acceptable vehicles and solvents arewater, Ringer's solution, phosphate buffered saline and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed mineral or non-mineral oil may be employed including syntheticmono-ordi-glycerides. In addition, fatty acids such as oleic acid finduse in the preparation of injectables. Carrier formulations suitable forsubcutaneous, intramuscular, intraperitoneal, intravenous, etc.administrations may be found in Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.

The adjuvants or antigens useful in the invention may be delivered inmixtures of more than two adjuvants or antigens. A mixture may consistof several adjuvants in addition to the synergistic combination ofadjuvants or several antigens.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular adjuvants orantigen selected, the age and general health status of the subject, theparticular condition being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of an immuneresponse without causing clinically unacceptable adverse effects.Preferred modes of administration are discussed above.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di-and tri-glycerides; hydrogelrelease systems; sylastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the invention iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

The use of CpG ODN as an adjuvant alone or in combination with otheradjuvants was evaluated. The hepatitis B virus surface antigen (HBsAg)given as a recombinant protein or expressed in vivo from a DNA vaccinewas used as an exemplary model system in the Examples set forth below.

Materials and Methods

Animals

Experiments on adult mice were carried out using female BALB/c mice(Charles River, Montreal, QC) at 6-8 weeks of age.

Newborn mice were obtained through breeding male and female BALB/c mice(Charles River) in the Loeb animal facility (Loeb Health ResearchInstitute, The Ottawa Hospital, Ottawa, ON). Pregnant females weremonitored daily to ensure accurate recording of the date of birth. Bothmale and female neonates were used for immunization.

Cynomolgus monkeys (1.5-3 kg) were housed at the Primate ResearchCenter, Bogor, Indonesia.

Orantutans (5-20kg) were housed at Wanariset Station for the OrangutanReintroduction Program of the Indonesian government, Balikpapan,Kalimantan.

HBsAg Subunit Vaccination of Mice

The subunit vaccine consisted of HBsAg (ay subtype) which had beenproduced as a recombinant protein in yeast cells (Medix Biotech#ABH0905). This was diluted in saline for use without adjuvant. HBsAgwas also formulated with alum and/or CpG ODN as adjuvant. HBsAg proteinwas mixed with aluminum hydroxide (Alhydrogel 85, [Al₂O₃], SuperfosBiosector, Vedbaek, Denmark) in the same ratio of 25 mg Al³⁺ per mgprotein as used in the commercial vaccines (i.e., 2.5 l 2% Al₂O₃ per μgHBsAg). The protein and alum were mixed with a vortex and then left onice for at least 30 minutes prior to use to allow the protein to adsorbonto the Al₂O₃. This solution was mixed again immediately prior toinjection by drawing up into the syringe 3-5 times.

For groups treated with CpG ODN, an appropriate volume of syntheticoligodeoxynucleotide (ODN #1826) of the sequence TCCATGACGTTCCTGACGTT(SEQ ID NO. 86) synthesized with a phosphorothioate backbone (OligosEtc. & Oligo Therapeutics, Wilsonville, Oreg.) was added alone or withalum to HBsAg on the day of injection. Adult mice received a singleintramuscular (IM) injection into the left tibialis anterior (TA) muscleof 1 or 2 ug HBsAg, without or with adjuvant (alum and/or CpG ODN), in50 l vehicle. When CpG DNA was added, each animal received a total of 1,10, 100 or 500 μg ODN. Newborn mice were immunized within 24 hours ofbirth or 7 days after birth by bilateral injection of a total of 1 μgHBsAg into the posterior thigh muscles (2×10 l @ 0.05 mg/ml). Allinjections were carried out with a 0.3 ml insulin syringe which has afused 29G needle (Becton Dickenson, Franklin Lakes, N.J.). For injectionof adults, the needle was fitted with a collar of polyethylene (PE)tubing to limit penetration of the needle to about 3 mm. Allintramuscular injections were carried out through the skin (shaved foradults) and under general anesthesia (Halothane, HalocarbonLaboratories, River Edge, N.J.).

HBsAg Subunit Vaccination of Monkeys

Monkeys were immunized by IM injection into the anterior thigh muscle ofEngerix-B® (SmithKline Beecham Biologicals, Rixensart, BE) whichcomprises HBsAg (ay subtype, 20 μg/ml) adsorbed to alum (25 mg Al3+/mgHbsAg). Each monkey received an injection of 0.5 ml containing 10 μgHbsAg. For some monkeys, 500 μg CpG ODN 1968 (TCGTCGCTGTTGTCGTTTCTT)(SEQ ID NO 72) was added to the vaccine formulation.

HBsAg Subunit Vaccination of Orangutans

Orangutans were immunized by IM injection into the anterior thigh muscleof HBsAg *ay subtype, 20 μg/ml) combined with alum (25 mg Al3+/mgHBsAg), combined with CpG. CpG ODN 2006 (TCGTCGTTTTGTCGTTTTGTCGTT) (SEQID NO 77) was added to the vaccine formulation. Each orangutan receivedan injection of 1.0 ml containing 20 μg HBsAg with alum (500 μg), CpGoligonucleotide (1 mg) or both adjuvants.

Experimental Groups

Comparison of CpG ODN and Non-nucleic Acid Adjuvants with HBsAg SubunitVaccine

Twelve groups of adult BALB/c mice (n=10) were injected with 1 μg HBsAg(i) alone, (ii) mixed with alum, (iii, iv, v, vi, vii) mixed with 0.1,1, 10, 100 or 500 μg CpG ODN, or (viii, ix, x, xi, xii) mixed with bothalum and 0.1, 1, 10, 100 or 500 μg CpG ODN. These mice were bled at 1,2, 4 and 8 weeks after immunization and the plasma was assayed foranti-HBs. At the end of the study the mice were killed and their spleensremoved for assay of CTL activity.

Other groups of mice (n=5) were immunized with HBsAg (1 μg) alone, withalum (25 μg Al3+), with one of several different CpG and non-CpG controloligonucleotides of different backbones (10 μg), or with both alum andan oligonucleotide.

Other groups of mice (n=5) were immunized as above (except only the 10μg dose of CpG ODN was used) and boosted with the identical or adifferent formulation at 8 weeks, then spleens were removed 2 weekslater for evaluation of CTL activity.

Other groups of mice were immunized with HBsAg (1 μg) and one of thefollowing non-nucleic acid adjuvants alone or in combination with CpGODN (10 μg): monophosphoryl lipid A (MPL, 50 μg, Ribi); Freund'sComplete Adjuvant (CFA; 1:1 v/v); Freund's Incomplete Adjuvant (IFA; 1:1v/v).

Immunization of Neonates with Subunit or DNA Vaccine

Groups of newborn and young BALB/c mice (n=10) aged <24 hours, 3, 7 or14 days were injected with (i, ii, iii) a total of 1 μg HBsAg with alum,with CpG ODN 1826 (10 μg) or with both alum and CpG ODN, or with (iv) anHBsAg-expressing DNA vaccine (1-μg pCMV-S). Plasma was obtained at 4, 8,12 and 16 weeks for assay of anti-HBs as total IgG and IgG subtypes(IgG1 and IgG2a). At the end of the study the mice were killed and theirspleens removed for assay of CTL activity.

Immunization of Cynomolgus Monkeys with HBsAg and Alum or Alum+CpG ODN

Two groups of juvenile Cynomolgus monkeys (n=5) were immunized at 0 and10 weeks with 0.5 ml Engerix-B (HBsAg at 20 mg/ml adsorbed to alum, 25mg Al3+/mg HBsAg) to which had been added saline (0.1 ml) or CpG ODN2006 (500 μg in 0.1 ml, SEQ #77). Monkeys were bled at 2, 8, 10, 12 and14 weeks and plasma was evaluated for anti-HBs titers (mIU/ml).

Immunization of Orangutans with HBsAg and Alum or CpG ODN or Alum+CpGODN

Three groups of juvenile orangutans were immunized IM at 0 and 4 weekswith 1 ml of vaccine containing HBsAg (10 μg) plus (i) alum (25 mgAl3+/mg HBsAg) (n=13), (ii) CpG ODN 2006 (SEQ# 77) (m=24) or (iii) alumplus CpG ODN (n=14). Animals were bled at 4.8 and 12 weeks and plasmawas evaluated for anti-HBs titers (mIU/ml).

Evaluation of Humoral Response to HbsAg

Mice: Heparinized blood was collected by retrobulbar puncture of lightlyanaesthetized mice as described elsewhere (Michel et al., 1995). Plasmawas recovered by centrifugation (7 min @ 13,000 rpm). Antibodiesspecific to HBsAg in plasma were detected and quantified by end-pointdilution ELISA assay (in triplicate) on individual samples. Ten-foldserial dilutions of plasma were first added to 96-well microtiter plateswith a solid phase consisting of plasma-derived HBsAg particles (100l/well of HBsAg ay subtype at 1 g/ml, coated overnight at RT) andincubated for 1 hr at 37C. The bound antibodies were then detected byincubation for 1 hr at 37C with HRP-conjugated goat anti-mouse IgG, IgM,IgG1 or IgG2a (1:4000 in PBS-Tween, 10% FCS; 100 l/well, SouthernBiotechnology Inc., Birmingham, Ala.), followed by incubation with OPDsolution (100 l/well, Sigma, St. Louis, Mo.) for 30 minutes at RT in thedark. The reaction was stopped by the addition of sulfuric acid (50 l of4N H₂SO₄). End-point titers were defined as the highest plasma dilutionthat resulted in an absorbance value (OD 450) two times greater thanthat of non-immune plasma with a cut-off value of 0.05. Anti-HBs titerswere expressed as group means of individual animal values, which werethemselves the average of triplicate assays.

Primates: Monkeys and orangutans were bled by antecubital venouspuncture into heparinized tubes and plasma was recovered bycentrifugation (7 min @ 13,000 rpm). Plasma was then evaluated foranti-HBs titers using a commercial kit (Monolisa anti-HBs,Pasteur-Sanofi) and expressed in milli-Intemational Units per milliliter(mIU/ml) by comparison with standards defined by the World HealthOrganization. A titer of 10 mIU/ml is considered sufficient to conferprotection to humans and great apes against infection by HBV.

Evaluation of Cytotoxic T Cell Response to HBsAg in Mice

Cytotoxic T-lymphocyte (CTL) activity was determined using splenocytestaken from mice 4 or 8 weeks post-prime or post-boost. In brief, singlecell suspensions were prepared and suspended in tissue culture medium(RPMI 1640, 10% FBS, Life Technologies, Grand Island, N.Y., supplementedwith 5×10⁻⁵ M β-mercaptoethanol and penicillinstreptomycin solution,1000 U/ml, 1 mg/ml final concentrations respectively, Sigma, and 3% El-4supematant as a source of IL-2). Splenocytes (3×10⁷) were co-culturedfor 5 days (37° C., 5% CO2) with 1×10⁶ syngenic HBsAg-expressingstimulator cells (P815S) or control target cells (P815) in round bottom96-well culture plates (37° C., 5% CO2, 4 hr). Supernatant (100 μl) wasremoved for radiation (gamma) counting. Spontaneous release wasdetermined by incubating target cells without effector cells and totalrelease by addition of 100 μl 2 N HC1 to the target cells. The percentlysis was calculated as [(experimental release−spontaneousrelease)/(total release−spontaneous release)]×100. The percent specificlysis was calculated as % lysis with P815S- % lysis with P815 cells. CTLactivity for responding mice [% specific lysis>10] were expressed asmeans ±SEM of individual animal values, which were themselves theaverage of triplicate assays.

Example 1 Synergy of CpG ODN with Alum as Adjuvant for HBV SubunitVaccine in Mice

A. Strength and Kinetics of Humoral Response

Immunization of BALB/c mice with HBsAg alone elicited only low titers ofanti-HBs (<100) by 4 weeks. These titers were about 10-fold higher withthe addition of alum as adjuvant, 60-fold higher with CpG ODN and morethan 500-fold higher with both alum and CpG ODN. At later time points,the highest peak titers were with HBsAg/alum/CpG, the second highestwith HBsAg/CpG, then HBsAg/alum (FIG. 1).

Similar synergistic results for antibody responses were obtained whenimmunization against HBsAg was carried out in neonatal and very youngmice, in which the immune system is immature. In mice immunized at 3days of age, where the immune system is even less mature than a newbornhuman, 10% and 0% of mice seroconverted with alum and CpG ODN alonerespectively, but 75% serocoinverted when CpG ODN and alum were usedtogether. In 7 day old mice, which have an immune system similar inmaturity to that of a newborn human, seroconversion for alum, CpG or thecombination was 11%, 22% and 100% respectively (FIG. 8). Furthermore, inthese 7 day old mice, antibody titers were up to 80-fold higher with thecombined adjuvants than with either adjuvant alone (FIG. 9).

When used alone or combined with alum, there is a dose-response for CpGODN with the best results being obtained with an intermediate dose (10μg) and no further or only relatively small gains with higher doses (upto 500 μg) when used alone or combined with alum respectively (FIG. 2).

When a large panel of ODN is compared for adjuvant activity it can beseen that CPG ODN with a nuclease-resistant phosphorothioate backbonehave the best adjuvant effects (FIG. 3). There was very little or noadjuvant activity of non-CpG control ODN with a phosphorothioatebackbone, or of CpG ODN with a chimeric or phosphodiester backbone.However, for those phosphorothioate CpG ODN that did not have adjuvanteffect, all exhibited a synergistic effect with alum. In general,antibody titers with combined alum and CpG ODN were 10 to 100-foldhigher than with CpG ODN and/or 100 to 1000-fold higher than with alumalone (FIG. 3).

B. Strength of Cytotoxic T-lymphocyte Response

CTL were very weak with HBsAg and no adjuvant, and were completely lostwith the addition of alum. CTL were augmented equally with both CpG ODNas with combined alum and CpG ODN (FIG. 1). A synergy for CTL responsescould be seen with prime-boost strategies, in that priming with CpG ODNand boosting with alum gave better CTL than priming and boosting withCpG alone (FIG. 4) (Note: use of alum alone completely abrogates the CTLresponse).

A synergistic action of CpG ODN and alum on CTL was very evident withimmunization of young (7 day old) mice. In this case, neither alum norCpG ODN used alone induced significant levels of HBsAg-specific CTL, butwhen used together there wre very strong CTL were observed (FIG. 9).

Thus, CpG ODN is superior to alum for both humoral and cell-mediatedresponses, when each is used alone as adjuvant with the HBsAg subunitvaccine in mice. When used together, there is a synergy of action suchthat antibody and CTL activity are stronger than when either adjuvant isused alone. These results indicate that CpG ODN could be used to replacealum in vaccine formulations, which could be desirable to avoidassociated side-effects due to local irritation in the muscle, or forcertain live-attenuated or multivalent vaccines where it is not possibleto use alum because chemical interactions interfere with the efficacy ofthe vaccine. This should not occur with CpG ODN. Of even greaterinterest is the strong synergistic response when CpG ODN and alum areused together as adjuvants. This could allow better immune responseswith lower or fewer doses of antigen. There is a fairly flat doseresponse to CpG ODN whether or not alum is present, indicating that awide range of CpG ODN could be useful to adjuvant vaccines in humans.

Example 2 Synergy of CpG ODN with Other Non-Nucleic Acid Adjuvants forHBV Subunit Vaccine in Mice

As discussed above, CpG ODN alone gave 8-fold higher antibody titersthan alum, the only adjuvant currently licensed for human use. It alsoproduces superior results to monphosphoryl lipid A (MPL, RibiPharmaceuticals, Middleton, WI), a new adjuvant that is currently inhuman clinical trials even when administered in a dose of five timesless than that of MPL. There was, as discussed above, a strong synergywith CpG ODN and alum, but in contrast no such synergy was seen with MPLand alum. Owing to the strong synergistic effect of alum and CpG ODN,this combination of adjuvants is even better than Freund's completeadjuvant (FCA) for inducing antibodies in mice (FIG. 5) Freund's, whichis considered the gold standard adjuvant for animal models is much tootoxic to use in humans.

The synergy seen with CpG ODN and alum, was also seen with CpG ODNcombined with other adjuvants. When used alone, CpG ODN and Freund'sincomplete adjuvant (FIA, a type of mineral oil) induced similarantibody titers, but when used together the anti-HBs titers were morethan 50-fold higher than with either adjuvant alone. Indeed, thecombination of CpG ODN and FIA was even better than FCA (FIG. 6).

Similarly, CpG ODN and MPL alone gave equally high antibody titers, butwhen used together the titers were about 4-times higher than with eitheradjuvant alone (FIG. 7). While the synergistic response with CpG and MPLwas not as marked with respect to overall antibody titers, it was verypronounced with respect to the Th1-bias of these antibodies (see below).

Example 3 Dominance and Synergy of CpG ODN with Alum for Induction of aTh1-Type Immune Response Including CTL

Immunization with either HBsAg alone or with alum induces apredominantly Th2-type humoral response with virtually no IgG2aantibodies, which are induced in response to Th1-type cytokines such asIL-12 and IFN-γ. Rather, almost all (>99%) antibodies were of the IgG1isotype IgG2a:IgG1=0.01. CpG ODN induces significantly more IgG2aantibodies, such that they made up at least 50% of the total IgG (IgG(IgG2a:IgG1=1.4). The combination of alum and CpG ODN induce an equallystrong Th1 response as CpG ODN alone (IgG2a:IgG1=1.0), despite theextremely strong Th2-bias of alum (FIG. 5). Similarly CTL responses withCpG ODN plus alum were as strong as those with CpG ODN alone, despitethe fact that the Th2-bias of alum resulted in a complete loss of CTLwhen alum was used alone (FIG. 1).

The strong Th1 bias with CpG is even more evident in neonatal and youngmice, which are known to naturally have a strong Th2-bias to theirimmune system. In this case, neither alum nor CpG ODN on their owninduced detectable IgG2a, indicating a very poor or absent Th1 response.Remarkably, when used together, CpG ODN and alum induced high levels ofIgG2a antibodies, which were now the predominant form of IgG (FIG. 10).Similarly, neither CpG ODN or alum induced significant levels of CTL inyoung mice, yet when used together there was a strong CTL response, thatwas even stronger than obtained with a DNA vaccine (FIG. 9).

The strength of the Th1 influence of CpG ODN is seen not only by itsability to dominate over the Th2 effect of alum when they areco-administered, but also to induce Th1 responses in animals previouslyprimed for a Th2 response with alum. Immunization with HBsAg using alumas an adjuvant completely abrogates the CTL response owing to the strongTh2 bias of alum (FIGS. 1 and 4). However, in mice using alum at primeand CpG at boost, good CTL were induced, indicating the possibility ofCpG to overcome a previously established Th2 response (FIG. 4).

Aluminum hydroxide (alum) is currently the only adjuvant approved forhuman use. An important disadvantage of alum is that it induces aTh2-rather than a Th1-type immune response, and this may interfere withinduction of CTL. Indeed, in mice immunized with recombinant HBsAg, theaddition of alum selectively blocked activation of CD8+ CTL (Schirmbecket al., 1994). Although not essential for protective immunity againstHBV, CTL may nevertheless play an important role. For example, a lack ofHBV-specific CTL is thought to contribute to the chronic carrier state.In contrast, one of the primary advantages of CpG DNA over alum as anadjuvant is the Th1-bias of the responses and thus the possibility toinduce CTL. A striking finding from the present study is that CpG cancompletely counteract the Th2-bias of alum when the two adjuvants aredelivered together, and in the case of immunization in early life, thecombination can even give a more Th1 response than CpG ODN alone. Thiscould allow one to capitalize on the strong synergistic action of thetwo adjuvants on the humoral response while still allowing CTL inadults, and to induce a stronger Th1 response in infants.

The use of alum has been linked to Th2-type diseases. The much higherprevalence of asthma (another Th2-type disease) in more highly developednations may be linked to the high hygiene level and rapid treatment ofchildhood infections (Cookson and Moffatt, 1997). Early exposure tobacterial DNA (and immunostimulatory CpG motifs) pushes the immunesystem away from Th2- and towards a Th1-type response and this mayaccount for the lower incidence of asthma in less developed countries,where there is a much higher frequency of upper respiratory infectionsduring childhood. Addition of CpG ODN as adjuvant to all pediatricvaccines could re-establish a Th1-type response thereby reducing theincidence of asthma.

Example 4 Synergy of CpG ODN with Other Adjuvants for Induction of aTh1-type Immune Responses

The synergistic effect of CpG ODN on Th1 responses was also seen usingother adjuvants. IFA on its own induces a very strong Th2-type responsewith virtually no IgG2a antibodies (IgG2a:IgG1=0.002) and CpG ODN on itsown induces a moderate Th1 response (IgG2a:IgG1=1.4), but together theresponse was very strongly Th1 (IgG2a:IgG1=24.0). It is notable thatthis is even more Th1 than the response induced by CFA (ratio=0.5) (FIG.6).

Similarly, CpG and MPL on their own are moderately Th1 (IgG2a:IgG1ratios at 4 weeks are 1.4 and 1.9 respectively), but together are verystrongly Th1 with a large predominance of IgG2a antibodies(ratio=83.3)(FIG. 7).

Example 5 CpG ODN as Synergistic Adjuvant in Cynomolgus Monkeys

CpG ODN, in combination with alum, also acts as a potent adjuvant toaugment anti-HBs responses in Cynomolgus monkeys. Compared to responsesobtained with the commercial HBV vaccine that contains alum, monkeysimmunized with the commercial vaccine plus CpG ODN attained titers50-times higher after prime and 10-times higher after boost (FIG. 14).

Example 6 CpG ODN as Synergistic Adjuvant to HBsAg in HyporesponderOrangutans

The orangutans, a great ape very closely related to man, can benaturally infected with HBV in a manner similar to humans.Unfortunately, orangutans are hyporesponsive to the commercial HBVvaccine that contains alum as an adjuvant. Compared to humans, where 13%and 56% of vaccinated individuals seroconvert by 4 weeks after first andsecond doses respectively (Yano and Tashiro, 1988), only 0% and 15% ofvaccinated orangutans have seroconverted by the same times. With theaddition of 1 mg CpG ODN, this becomes 43% and 100% respectively. Asynergistic response is seen even in these hyporesponders, becauseantibody levels and seroconversion rates are better with CpG ODN plusalum than with either adjuvant alone (FIG. 12).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

REFERENCES

-   1. Chen D. S. et al. (1996). Cancer Causes & Control 7: 305-311.-   2. Cookson, W. O. C. M. & Moffatt, M. F. (1997). Science 275: 41-42.-   3. Cowdery, J. S. et al. (1996). J. Immunol. 156: 4570.-   4. Davis, H. L. et al. (1993). Human Molec. Genet. 2: 1847-1851.-   5. Davis, H. L. et al. (1995). Human Gene Ther. 6:1447-1456.-   6. Davis, H. L. et al. (1996). Proc. Natl. Acad. Sci. USA 93:    7213-7218.-   7. Davis, H. L. et al. (1996). Vaccine 14: 910-915.-   8. Davis, H. L. et al. (1997). Gene Ther. (in press).-   9. Davis, H. L. & Brazolot Millan, C. L. (1997). Blood Cell Biochem.    (in press).-   10. Donnelly, J. J. et al. (1996). Life Sciences. 60: 163-172.-   11. Dubois, M. -F. et al. (1980). Proc. Natl. Acad. Sci. USA 77:    4549-4553.-   12. Ellis, R. W. (Ed.) (1993). Hepatitis B Vaccines in Clinical    Practise. New York: Marcel-Dekker.-   13. Halpern, M. D. et al. (1996). Cell. Immunol. 167: 72.-   14. Klinman, D. M. et al (1996). Proc. Natl. Acad. Sci. USA 93:    2879-2883.-   15. Krieg, A. M. et al. (1995). Nature 374: 546-549.-   16. Kruskall, M. S. et al. (1992). J. Exp. Med. 175: 495-502.-   17. Lee C. Y. et al., (1989). J. Am. Med. Assoc. SEA-   18. Li et al. , (1994). J. Gen. Virol. 75: 3673-3677.-   19. Mancini, M. etal. (1996). Proc. Natl. Acad. Sci. USA 93:    12496-12501.-   20. Michel, M.-L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:    7708-7712.-   21. Michel, M.-L. et al. (1995). Proc. Natl. Acad. Sci. USA 92:    5307-5311.-   22. Milich, D. R. (1988). Immunol. Today 9: 380-386.-   23. Niederau etal. (1996). New Eng. J. Med. 334:1422-1427.-   24. Pol, S. etal. (1993). C. R. Acad. Sci. (Paris) 316: 688-691.-   25. Rehermann, B. et al. (1996). Nature Med. 2: 1104-1108.-   26. Sato, Y., etal. (1996). Science 273: 352-354.-   27. Schirmbeck, R. et al. (1994). J. Immunol. 152: 1110-1119.-   28. Stevens, C. E. et al. (1987). J. Am. Med. Assoc. 257: 2612-2616.-   29. Vogel, F. R. & Sarver, N. (1995). Clin. Microbiol. Rev. 8:    406-410.-   30. Yano, M and Tashiro, A. (1988) In: Viral Hepatitis and Liver    Disease, Ed: A. J. Zuckerman; Alan R. Liss, New York, pp 1038-1042.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A method of inducing an antigen specific immune response in asubject, comprising: administering to the subject in order to induce anantigen specific immune response an antigen and a combination ofadjuvants, wherein the combination of adjuvants includes at least oneoligonucleotide containing at least one unmethylated CpG dinucleotideand at least one non-nucleic acid adjuvant, wherein the non-nucleic acidadjuvant is an immune stimulating adjuvant selected from the groupconsisting of PCPP polymer, derivatives of lipopolysaccharides, MPL,MDP, t-MDP, OM-174 and Leishmania elongation factor, wherein thecombination of adjuvants is administered in an effective amount forinducing a synergistic adjuvant response, and wherein theoligonucleotide is 8-100 nucleotides in length and has at least onephosphorothioate backbone modification.
 2. The method of claim 1,wherein the combination of adjuvants is administered with a priming doseof antigen.
 3. The method of claim 1, wherein the combination ofadjuvants is administered with a boost dose of antigen.
 4. The method ofclaim 2, wherein the subject is administered a boost dose of antigen andoligonucleotide containing at least one unmethylated CpG dinucleotideafter the priming dose.
 5. The method of claim 3, wherein the subject isadministered a priming dose of antigen and oligonucleotide containing atleast one unmethylated CpG dinucleotide before the boost dose.
 6. Themethod of claim 1, wherein the oligonucleotide containing at least oneunmethylated CpG dinucleotide has a sequence including at least thefollowing formula:5′ X₁X₂CGX₃X₄ 3′ wherein C and G are unmethylated, wherein X₁X₂ and X₃X₄are nucleotides.
 7. The method of claim 6, wherein the 5′ X₁X₂CGX₃X₄ 3′sequence is a non-palindromic sequence.
 8. The method of claim 6,wherein X₁X₂ are nucleotides selected from the group consisting of: GpT,GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG; and X₃X₄ arenucleotides selected from the group consisting of: TpT, CpT, ApT, TpG,ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA.
 9. The method of claim 6,wherein X₁X₂ are selected from the group consisting of GpA and GpT andX₃X₄ are TpT.
 10. The method of claim 6, wherein X₁X₂ are both purinesand X₃X₄ are both pyrimidines.
 11. The method of claim 6, wherein X₂ isa T and X₃ is a pyrimidine.
 12. The method of claim 6, wherein theoligonucleotide is 8 to 40 nucleotides in length.
 13. The method ofclaim 6, wherein the oligonucleotide is isolated.
 14. The method ofclaim 6, wherein the oligonucleotide is a synthetic oligonucleotide. 15.The method of claim 1, wherein the subject is an infant.
 16. The methodof claim 1, wherein the antigen is derived from an infectious organismselected from the group consisting of a virus, bacterium, fungus andparasite.
 17. The method of claim 1, wherein the antigen is a tumorantigen.
 18. The method of claim 1, wherein the antigen is an allergen.19. The method of claim 1, wherein the antigen is in the form of a crudeextract.
 20. The method of claim 1, wherein the antigen is in the formof a purified molecule including a protein or a polysaccharide.
 21. Themethod of claim 1, wherein the antigen is in the form of a recombinantmolecule including a protein, polypeptide, peptide or peptide mimic of apolysaccharide antigen.
 22. The method of claim 1, wherein thenon-nucleic acid adjuvant by itself gives a Th1 immune response (e.g.,MPL) but when used in combination with the CpG oligonucleotide gives astronger Th1 response.